Methods and compositions for diagnosing and treating cancer

ABSTRACT

The invention provides assays, methods, systems, compositions, and kits for diagnosing and treating cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 Natlonal Phase Entry Application ofInternational Application No. PCT/US2012/072264 filed Dec. 31, 2012,which designates the U.S., and which claims benefit under 35 U.S.C.§119(e) of the U.S. Provisional Application No. 61/581,317, filed Dec.29, 2011, the content of which is incorporated herein by reference inits entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 10, 2013, isnamed 04321407.txt and is 45,023 bytes in size.

TECHNICAL FIELD

The present disclosure relates generally to assays, methods, systems andcompositions for diagnosing and treating cancer in a subject.

BACKGROUND

The 14-3-3 protein family includes seven highly conserved dimericisoforms (β, γ, ε, ζ, η, σ, and τ) that are expressed in all eukaryoticcells (1). Through interaction with phospho-serine or phospho-threoninemotifs, 14-3-3 can regulate diverse cellular functions, including signaltransduction, cytoskeletal configuration, metabolism, differentiation,survival, and transcription (2). 14-3-3 proteins are implicated intumorigenesis (3, 4), as a tumor suppressor in the case of 14-3-3σ(SFN), and as a putative oncoprotein in the case of 14-3-3ζ (YWHAZ).14-3-3σ expression is inhibited in premalignant and malignant cells (5),and loss of 14-3-3σ results in polyploidy and failure to maintain G2/Mcell-cycle arrest after DNA damage through cytoplasmic sequestration ofCDC2/cyclin B1 (6, 7). 14-3-3ζ expression is up-regulated in variouscancers (8), and it induces epithelial-mesenchymal transition byactivation of TGF-β/Smads and inhibits apoptosis in anoikic cells,thereby potentiating tumor invasion and metastasis (9, 10).

Endometrial stromal sarcoma (ESS) is a type of uterine sarcoma that, inits low-grade form, contains JAM fusions with various polycomb complexproteins (SUZ12, PHF1, and EPC1) (11, 12). In contrast, some ESS arehistologically high grade, and these tumors typically lack JAZF1rearrangement. The genetic basis for high-grade ESS is undefined.

SUMMARY

The present invention is based, in part, on inventors' discovery of atransforming 14-3-3 oncoprotein. 14-3-3 proteins are ubiquitouslyexpressed regulators of various cellular functions, includingproliferation, metabolism, and differentiation, and altered 14-3-3expression is associated with development and progression of cancer. Theinventors used a combination of cytogenetics and next-generationsequencing to identify YWHAE-FAM22A/B genetic fusion as a frequentgenetic event that is specific for high-grade ESS. The inventors furtherdemonstrated the transforming properties of the fusion protein andcharacterized the clinicopathologic significance of YWHAE-FAM22A/Bgenetic fusion. The discovery of this unique oncogenic mechanism hasbiologic, diagnostic, and therapeutic implications.

Accordingly, in one aspect provided herein is a method of identifying asubject suitable for endometrial stromal sarcoma (ESS) treatment.Generally, the method comprises detecting the presence of a YWHAE-FAM22fusion protein or a nucleic acid encoding the same in a biologicalsample taken from the subject. Presence of the fusion protein or thenucleic acid encoding the fusion protein indicating that the individualshould undergo anti-cancer treatment, e.g., treatment for endometrialstromal sarcoma.

After a subject is identified as needing anti-cancer treatment, thesubject can be treated with an anti-cancer treatment. Thus, in anotheraspect provided herein is method of treating endometrial stromal sarcomain subject in need thereof, the method comprising administering ananti-cancer therapy to the subject, wherein the subject expresses aYWHAE-FAM22 fusion protein or a nucleic acid encoding the same. In someembodiments, the method comprising: assaying a biological sample from asubject for presence of the YWHAE-FAM22 fusion protein or a nucleic acidencoding the same and administering an anti-cancer therapy to thesubject if the YWHAE-FAM22 fusion protein or a nucleic acid encoding thesame is detected in the sample.

Also provided herein is an isolated sample from a subject, wherein thesample comprises a YWHAE-FAM22 fusion protein or a nucleic acid encodingthe same. In some embodiments, the sample further comprises a firstreagent that can bind with the fusion protein or the nucleic acid.

The invention also provides an isolated nucleic acid encoding theYWHAE-FAM22 fusion protein described herein. Additionally, the inventionalso provides an isolated YWHAE-FAM22 fusion protein. In someembodiments, the YWHAE-FAM22 fusion protein or the nucleic acid encodingthe same is from a biological sample taken from a subject.

Further provided herein is a composition, comprising: (i) a YWHAE-FAM22fusion protein or a nucleic acid encoding the same; and (ii) a reagentthat binds with the fusion protein or the nucleic acids. In someembodiments, the reagent is adapted to produce a signal so as to detectpresence of the fusion protein or the nucleic acid in the sample.

Without limitations, the FAM22 protein portion of the fusion protein canbe any FAm22 family member, such as FAM22A, FAM22B, FAM22C, FAM22D orFAM22E. In embodiments of the various aspects described herein, theYWHAE-FAM22 fusion protein can be a YWHAE-FAM22A or YWHAE-FAM22B fusionprotein, or can be a YWHAE-FAM22 fusion involving other FAM22 familymembers, including FAM22C, FAM22D and FAM22E.

In embodiments of the various aspects described herein, the nucleic acidencoding the YWHAE-FAM22 fusion protein comprises the nucleotidesequence SEQ ID NO:1 or SEQ ID No: 2.

In embodiments of the various aspects described herein, the YWHAE-FAM22fusion protein comprises the amino acid sequence SEQ ID NO: 3 or SEQ IDNO: 4.

In some embodiments, the biological sample comprises endometrial cells.

Also provided herein is a detection assay, the assay comprisingdetecting presence of a YWHAE-FAM22 fusion protein or a nucleic acidencoding the same in a biological sample taken from a subject. In someembodiments, the subject is suspected of having a cancer.

The assays, methods, systems, compositions, and kits described hereincan be used for classifying endometrial cancer in a subject. Asdiscussed herein, subjects expressing the YWHAE-FAM22 fusion proteinhave uterine sarcomas that are genetically, histologically, andclinically distinct from other forms of uterine sarcomas.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show the genomic mechanisms for the 14-3-3 fusion oncogenein endometrial cancer. FIG. 1A, high-grade ESS G-banded partialkaryotype showing a balanced translocation, t(10;17). Arrows indicatethe translocation breakpoints. FIG. 1B, split-apart view ofYWHAE-flanking BACs, RP11-22012 (red) and RP11-100F18 (green),demonstrates YWHAE rearrangement in an ESS1 cell. FIG. 1C, deFuseanalysis of ESS1 whole-transcriptome paired-end sequencing (Illumina)identifies split-read transcript sequences in which YWHAE exon 5 isfused to FAM22A exon 2. The conserved 14-3-3 protein-binding domains areencoded by exons 2 and 4 of YWHAE (denoted by the lines on exons 2 and 4of YWHAE). Split-read nucleotide sequences are SEQ ID NO: 5 to SEQ IDNO: 80 in order of appearance (from top to bottom).

FIG. 1E, RT-PCR using YWHAE exon 1 (lanes 1 and 2) and exon 5 (lane 3)forward primers with FAM22A/B/E exon 2 reverse primer in twot(10;17)-bearing ESS. Sequence analyses showed YWHAE-FAM22A andYWHAE-FAM22B, respectively, in ESS1 and ESS3. The top, middle, andbottom arrows indicate 1,650-, 1,000-, and 650-Kb markers, respectively.FIG. 1E, schematic of YWHAE on chromosome 17 (Chr 17) and the twoalternative fusion partners, FAM22A and FAM22B on chromosome 10 (Chr10), with the direction of transcription indicated by arrows.

FIGS. 2A-2D show the oncogenic roles of YWHAE-FAM22A fusion oncoproteinand structural considerations. FIG. 2A, 3T3 cells transfected(Lipofectamine) with YWHAE-FAM22A pcDNA3 had increased cell viability(CellTiter Glo luminescence assay) at various plating densities comparedwith 3T3 cells transfected with YWHAE pcDNA3. Error bars indicate SEs.FIGS. 2B and 2C, 3T3 cells transfected (Lipofectamine) with YWHAE-FAM22ApcDNA3 migrated more rapidly than 3T3 cells transfected with YWHAEpcDNA3, as shown by assays for quantitative cell migration (FIG. 2B) andwound healing (FIG. 2C). Error bars indicate SEs. FIG. 2D, structuralmodeling of YWHAE-FAM22A (including the protein sequences encoded byexons 1 to ˜5 of YWHAE and FAM22A exon 2) based on the X-ray crystalstructure of 14-3-3. Heterodimer of YWHAE-FAM22 bound to native YWHAE isdepicted in stick representation. The green and cyan chains indicateYWHAE (14-3-3ε) sequences with the purple helix representing the firstpart of FAM22A. This model shows that YWHAE fusion to FAM22 is unlikelyto interfere with YWHAE dimerization or phosphopeptide binding.

FIGS. 3A and 3B show that oncogenic fusion to FAM22 enables aberrantnuclear localization of YWHAE. FIG. 3A, endogenous YWHAE-FAM22A ispredominantly nuclear, whereas native YWHAE is predominantlycytoplasmic. FOXO3A and poly(ADP-ribose) polymerase (PARP) are nuclearlocalization controls, whereas GAPDH is a cytoplasmic control. FIG. 3B,induced YWHAE-FAM22A expression is nuclear in 293T cells, as shown byFLAG immunoprecipitation (Upper) and YWHAE immunohistochemistry (Lower)after transient expression of FLAG-tagged YWHAE-FAM22A pcDNA3 construct.In contrast to the predominantly cytoplasmic staining (and absentnuclear staining) seen in nontransfected 293T cells (representingwild-type YWHAE), YWHAE immunostaining in YWHAE-FAM22A-expressing 293Tcells showed the presence of nuclear staining, indicating nuclearlocalization of the fusion protein.

FIGS. 4A-4D show that YWHAE-FAM22 ESS is associated with distinctivehistology, gene-expression profiles, and clinical behavior. FIG. 4A,YWHAE-FAM22 ESS, in contrast to JAZF1-SUZ12 ESS, has high-gradehistology, with larger and more irregular nuclei and increased mitoticactivity. FIG. 4B, 3′ sequencing gene-expression profiling withunsupervised hierarchical clustering demonstrates distinctgene-expression signatures between YWHAE-FAM22 ESS (YWHAE ESS),JAZF1-rearranged ESS (JAZF1 ESS), and uterine leiomyosarcoma (LMS). FIG.4C, patients with YWHAE-FAM22 ESS present with higher InternationalFederation of Gynecology and Obstetrics (FIGO) stage disease comparedwith patients with JAZF1-rearranged ESS. FIG. 4D, YWHAE-FAM22 ESS(average follow-up period of 3.5 y) more frequently recurs compared withJAZF1-rearranged ESS (average follow-up period of 10 y). NED, noevidence of disease; AWD, alive with disease; DOD, died of disease.

FIGS. 5A and 5B show results of FISH studies. FIG. 5A, FISH studiesusing BAC probes flanking the breakpoint region in 10q23.2 in ESS1 and10q22.3 in ESS12. The 10q breakpoints were mapped to a 725-Kb region(flanked by BACs RP11-1005L9 and RP11-210E13) in 10q23.2 and a 600-Kbregion (flanked by BACs RP11-715A21 and RP11-668E21) in 10q22.3. FIG. 5B(upper), whole-genome sequencing (20× coverage, blue trace) of a normalhuman DNA, demonstrating that the FISH-mapped ESS 10q22.3 and 10q23.2translocation breakpoints (red arrows) are in localized regions ofextremely poor sequence mappability. FIG. 5B (lower), depictshigher-resolution view of the 10q23.2 breakpoint region showinglocations of the FAM22A and FAM22D genes. A similar organization alsoapplies to the FAM22B and FAM22E genes in the 10q22.3 breakpoint region.

FIGS. 6A and 6B show YWHAE-FAM22 fusion protein expression. YWHAE-FAM22is expressed in high-grade cancers with t(10;17) (ESS1 and ESS3) but notin low-grade ESS JAZF1-SUZ12 (ESS-JAZF1), leiomyosarcoma (LMS), orgastrointestinal stromal tumor (GIST). The lane marked 293 p-YFAcontains 293T cells expressing FLAGtagged YWHAE-FAM22A pcDNA3 construct.YWHAE-FAM22 alternate forms (˜110 kDa and ˜140 kDa) were demonstrated byan N-terminal YWHAE antibody (HPA008445; Sigma; developed against apeptide containing amino acids 1 to ˜0.70 of YWHAE) but not by aC-terminal YWHAE antibody (BML-SA475; Enzo Life Sciences; developedagainst a peptide containing amino acids 239 to ˜255 of YWHAE, a regionencoded by YWHAE exon 6), whereas both N-terminal and C-terminal YWHAEantibodies identified the wild-type YWHAE (˜0.30 kDa). The N-terminalYWHAE antibody also identified other native 14-3-3 family proteins,represented by the lower bands (˜0.27 kDa).

FIG. 7A is an immunoblot showing inhibition of YWHAE-FAM22A fusionprotein expression in ESS1 infected with shRNA1 (targets exon 2 ofFAM22A), but not in ESS1 infected with empty lentiviral vector or shRNA2(targets exon 1 of FAM22A, which is not in the YWHAE-FAM22A fusiongene).

FIG. 7B shows representative images of ESS1 in monolayer culture showingreduced cell growth (at 15 d) in ESS1 infected with shRNA1 compared withlentiviral empty vector or shRNA2.

FIG. 7C shows results of a wound healing assay showing reduced cellmigration of ESS1 infected with shRNA1 compared with lentiviral emptyvector.

FIGS. 8A and 8B show that siRNA targeting exon 2 and exon 7 of FAM22A(siFAM22A) reduced YWHAE-FAM22A fusion protein expression (N-terminalYWHAE immunoblot (FIG. 8A) and cell viability (CellTiter Gloluminescence assay) (FIG. 8B) in ESS1 containing YWHAE-FAM22A (day 4after Lipofectamine transfection). Error bars indicate SEs.

FIG. 9 is a block diagram showing an exemplary system for use in themethods described here, e.g., for selecting subject for treatment forESS.

FIG. 10 is an exemplary set of instructions on a computer readablestorage medium for use with the systems described herein.

DETAILED DESCRIPTION

14-3-3 proteins are ubiquitously expressed regulators of variouscellular functions, including proliferation, metabolism, anddifferentiation, and altered 14-3-3 expression is associated withdevelopment and progression of cancer. The inventors have now discovereda transforming 14-3-3 oncoprotein. The inventors discovered thetransforming 14-3-3 oncoprotein through cytogenetics andwhole-transcriptome sequencing analysis as a highly recurrent geneticmechanism in a clinically aggressive form of uterine sarcoma: high-gradeendometrial stromal sarcoma (ESS). As described herein, the 14-3-3oncoprotein results from a t(10;17) genomic rearrangement, leading tofusion between 14-3-3ε (YWHAE) and either of two nearly identical FAM22family members (FAM22A or FAM22B). Expression of YWHAE-FAM22 fusiononcoproteins was demonstrated by immunoblot in t(10;17)-bearing frozentumor and cell line samples. YWHAE-FAM22 fusion gene knockdowns wereperformed with shRNAs and siRNAs targeting various FAM22A exons in ant(10;17)-bearing ESS cell line (ESS1): Fusion protein expression wasinhibited; with corresponding reduction in cell growth and migration.YWHAE-FAM22 maintains a structurally and functionally intact 14-3-3e(YWHAE) protein-binding domain, which is directed to the nucleus by aFAM22 nuclear localization sequence. In contrast to classic ESS,harboring JAZF1 genetic fusions, YWHAE-FAM22 ESS display high-gradehistologic features, a distinct gene-expression profile, and a moreaggressive clinical course. Fluorescence in situ hybridization analysisdemonstrated absolute specificity of YWHAE-FAM22A/B geneticrearrangement for high-grade ESS, with no fusions detected in otheruterine and nonuterine mesenchymal tumors (55 tumor types, n=827). Thesediscoveries reveal diagnostically and therapeutically relevant modelsfor characterizing aberrant 14-3-3 oncogenic functions. Based on theinventors' discovery of the transforming 14-3-3 oncoprotein, providedhere are methods and compositions for diagnosing and treating cancer,e.g., high-grade endometrial stromal sarcoma.

Accordingly, in one aspect, the invention relates to a method ofassessing endometrial stromal sarcoma status in a subject which involvesthe step of assaying a biological sample derived from the individual forthe presence of fusion protein or a nucleic acid encoding the fusionprotein. In particular, the invention relates to a method of assistingin clinical decision making during screening of a subject (e.g., apatient) for identifying subjects suitable for endometrial stromalsarcoma treatment, which involves the step of assaying a biologicalsample derived from the individual for the presence of a fusion proteinor a nucleic acid encoding the fusion protein. Presence of the fusionprotein or the nucleic acid encoding the fusion protein indicating thatthe individual should undergo anti-cancer treatment, e.g., treatment forendometrial stromal sarcoma. Without limitations, a fusion protein orthe nucleic acid can be detected in a sample directly, by assaying forthe fusion protein or the nucleic acid, or indirectly by assaying for areagent that binds with the fusion protein or the nucleic acid.Exemplary such methods are described herein below.

As used herein, the term “fusion protein” or grammatical equivalentsthereof is meant a protein composed of a plurality of polypeptidecomponents, that while typically unjoined in their native state, arejoined by their respective amino and carboxyl termini through a peptidelinkage to form a single continuous polypeptide. Fusion proteins can bea combination of two, three or even four or more different proteins.

In some embodiments, the fusion protein comprises a portion of YWHAEprotein and a portion of a FAM22 protein. Without limitations the FAM22protein can be any member of the FAM22 family. For example, the FAM22protein can be, but is not limited to, FAM22A, FAM22B, FAM22C, FAM22D,or FAM22E. In some embodiments, the FAM22 protein is FAM22A or FAM22Bprotein. In other embodiments, based on very high sequence conservationamong the FAM22 family members, the FAM22 protein is FAM22C, FAM22D, orFAM22E.

In some embodiments, the N-terminal of the fusion protein comprises aportion of the YWHAE protein and the C-terminal of the fusion proteincomprises a portion of the FAM22 protein.

In some embodiments, the nucleic acid encoding the fusion proteincomprises exon 5 of the gene encoding the YWHAE protein.

In some embodiments, the nucleic acid encoding the fusion proteincomprises exon 2 of the gene encoding a FAM22 (e.g., FAM22A or FAM22B)protein.

In some embodiments, in the nucleic acid encoding the fusion protein,exon 5 of the gene encoding the full length YWHAE protein is linked toexon 2 of the gene encoding the FAM22 protein.

In some embodiments, the nucleic acid encoding the YWHAE-FAM22 fusionprotein comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2.

In some embodiments, the fusion protein comprises the amino acidsequence of SEQ ID NO: 3 or SEQ ID NO: 4.

Accordingly, in one aspect provided herein is a method of identifying asubject suitable for endometrial stromal sarcoma treatment. The methodcomprises detecting the presence of a YWHAE-FAM22 fusion protein or anucleic acid encoding the same in a biological sample taken from thesubject. Presence of the fusion protein or the nucleic acid encoding thefusion protein indicating that the individual should undergo anti-cancertreatment, e.g., treatment for endometrial stromal sarcoma.

As used herein, the term “biological sample” refers to a sample obtainedfrom an organism or from components (e.g., cells) of an organism. Thesample can be of any biological tissue or fluid. Frequently the samplewill be a “clinical sample” which is a sample derived from a patient.Such samples include, but are not limited to, sputum, blood, blood cells(e.g., white cells), tissue or fine needle biopsy samples, urine,peritoneal fluid, and pleural fluid, or cells therefrom. Biologicalsample can be a respiratory sample, bone marrow aspirations,cerebrospinal fluid, urine or blood fluid. Blood fluid means blood,serum or plasma. Biological samples can also include sections of tissuessuch as frozen sections taken for histological purposes. The term“biological sample” also includes untreated or pre-treated (orpre-processed) biological samples.

In some embodiments, the biological sample can be a biological fluid,including, but not limited to, blood (including whole blood, plasma,cord blood and serum), lactation products (e.g., milk), amniotic fluids,sputum, saliva, urine, semen, cerebrospinal fluid, bronchial aspirate,perspiration, mucus, liquefied feces, synovial fluid, lymphatic fluid,tears, tracheal aspirate, and fractions thereof. In other embodiments,the biological sample can include cell lysate and fractions thereof. Forexample, cells (such as red blood cells, platelets, white blood cellsand any cells circulating in the biological fluid described herein) canbe harvested and lysed to obtain a cell lysate. In some embodiments, abiological sample is a blood sample. In some embodiments, a biologicalsample is a plasma sample. In some embodiments, a biological sample is asaliva sample. In some embodiments, a biological sample is a buccalsample. In some embodiments, a biological sample is a urine sample.

In some embodiments, the sample is from a resection, biopsy, or coreneedle biopsy. In addition, fine needle aspirate samples can be used.

In some embodiments, the biological sample comprises endometrial cells.The sample can be obtained by removing a sample of cells from a subject,but can also be accomplished by using previously isolated cells (e.g.isolated by another person). In addition, the biological sample can befreshly collected or a previously collected sample.

Samples can also be either paraffin-embedded or frozen tissue.Accordingly, in some embodiments, the biological sample can be a frozenbiological sample, e.g., a frozen tissue or fluid sample such as urine,blood, serum or plasma. The frozen sample can be thawed before employingthe methods, assays and systems described herein. After thawing, afrozen sample can be centrifuged before being subjected to methods,assays and systems described herein.

In some embodiments, the test sample or the biological sample can betreated with a chemical and/or biological reagent. Chemical and/orbiological reagents can be employed to protect and/or maintain thestability of the sample, including biomolecules (e.g., nucleic acid andprotein) therein, during processing. One exemplary reagent is a proteaseinhibitor, which is generally used to protect or maintain the stabilityof protein during processing. In addition, or alternatively, chemicaland/or biological reagents can be employed to release nucleic acid orprotein from the sample. The skilled artisan is well aware of methodsand processes for collecting and/or preprocessing of different types ofbiological samples.

Methods for assaying a biological sample for the presence a protein,e.g. a fusion protein are well known to those skilled in the art. Suchmethods include, but are not limited to, immunoassays that include, butare not limited to, immunoprecipitation assays, ELISA-based assays,radioimmunoassay, “sandwich” immunoassays, immunodiffusion assays,agglutination assays, and western blot assays. Similarly, methods forassaying a biological sample for the presence of a nucleic acid are alsowell known in those skilled in the art. Such methods include, but arenot limited to, restriction enzyme digestion, probe hybridization,primer extension, sequence specific amplification, sequencing, 5′nuclease digestion, molecular beacon assays, oligonucleotide ligationassays, and Northern Blot. Exemplary methods for detecting the fusionprotein or the nucleic acid encoding the same are described in detailbelow.

Without limitations, substantially any method of detecting a nucleicacid can be used in assaying a sample for presence of the nucleic acidencoding the fusion protein. Such methods, include, but are not limitedto, restriction enzyme digestion, probe hybridization, primer extension,sequence specific amplification, sequencing, 5′ nuclease digestion,molecular beacon assays, and oligonucleotide ligation assays.

In some embodiments, detection of the nucleic acid in the sample is byDNA sequencing. Exemplary DNA sequencing methods include, but are notlimited to, Maxam-Gilbert sequencing; Chain-termination methods;advanced methods and de novo sequencing, such as shotgun sequencing,bridge PCR, and the like), Next-generation methods, such as MassivelyParallel Signature Sequencing (MPSS), Polony sequencing, 454pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, Ionsemiconductor sequencing, DNA nanoball sequencing, Heliscope singlemolecule sequencing, Single molecule real time (SMRT) sequencing), andmethods such as Nanopore DNA sequencing, Sequencing by hybridization,Sequencing with mass spectrometry, Microfluidic Sanger sequencing,Microscopy-based techniques, RNAP sequencing, in vitro virushigh-throughput sequencing, and the like.

Methods for detecting nucleic acids can include the use of distinctoligonucleotide probes, for example oligonucleotides complementary to aportion of the nucleotide sequence of the nucleic acid of interest,e.g., a nucleic acid encoding the fusion protein. The probe ispreferably a DNA oligonucleotide having a length in the range from about20 to about 40 nucleotide residues, preferably from about 20 to about 30nucleotide residues, and more preferably having a length of about 25nucleotide residues.

In some embodiments, the probe is rendered incapable of extension by aPCR-catalyzing enzyme such as Taq polymerase, for example by having afluorescent probe attached at one or both ends thereof. Althoughnon-labeled oligonucleotide probes can be used in the kits and methodsdescribed herein, the probes are preferably detectably labeled.Exemplary labels include radionuclides, light-absorbing chemicalmoieties (e.g. dyes), fluorescent moieties, and the like. Preferably,the label is a fluorescent moiety, such as 6-carboxyfluorescein (FAM),6-carboxy-4,7,2′,7′-tetrachlorofluoroscein (TET), rhodamine, JOE(2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein), HEX(hexachloro-6-carboxyfluorescein), or VIC.

In some embodiments, the probe can comprise both a fluorescent label anda fluorescence-quenching moiety such as6-carboxy-N,N,N′,N′-tetramethylrhodamine (TAMRA), or4-(4′-dimethlyaminophenylazo)benzoic acid (DABCYL). When the fluorescentlabel and the fluorescence-quenching moiety are attached to the sameoligonucleotide and separated by no more than about 40 nucleotideresidues, and preferably by no more than about 30 nucleotide residues,the fluorescent intensity of the fluorescent label is diminished. Whenone or both of the fluorescent label and the fluorescence-quenchingmoiety are separated from the oligonucleotide, the intensity of thefluorescent label is no longer diminished. Preferably, the probe for usein the assays, methods, systems and kits described herein can have afluorescent label attached at or near (i.e. within about 10 nucleotideresidues of) one end of the probe and a fluorescence-quenching moietyattached at or near the other end. Degradation of the probe by aPCR-catalyzing enzyme releases at least one of the fluorescent label andthe fluorescence-quenching moiety from the probe, thereby discontinuingfluorescence quenching and increasing the detectable intensity of thefluorescent labels. Thus, cleavage of the probe (which, as discussedabove, is correlated with complete complementarity of the probe with thetarget portion) can be detected as an increase in fluorescence of theassay mixture.

If detectably different labels are used, more than one labeled probe canbe used. For example, the assay mixture can contain a first probe whichis complementary to a portion of the nucleic acid that encodes the YWHAEportion of the fusion protein and to which a first label is attached,and a second probe which is complementary to a portion of the nucleicacid that encodes the FAM22 portion of the fusion protein. When twoprobes are used, the probes are detectably different from each other,having, for example, detectably different size, absorbance, excitation,or emission spectra, radiative emission properties, or the like. Forexample, a first probe can have FAM and TAMRA attached at or nearopposite ends thereof. The first probe can be used in the methods,assays, systems and kits described herein together with a second probewhich has TET and TAMRA attached at or near opposite ends thereof.Fluorescent enhancement of FAM (i.e. effected by cessation offluorescence quenching upon degradation of the first probe by Taqpolymerase) can be detected at one wavelength (e.g. 518 nanometers), andfluorescent enhancement of TET (i.e. effected by cessation offluorescence quenching upon degradation of the second probe by Taqpolymerase) can be detected at a different wavelength (e.g. 582nanometers).

In some embodiments, the nucleic acid detection can comprise amplifyingthe target nucleic acid. This can be accomplished using a pair ofamplification primers for amplifying a reference region of the nucleicacid encoding the fusion protein. In some embodiments, the referenceregion comprises the a portion of the nucleic acid encoding the YWHAEportion of the fusion protein and a portion of the nucleic acid encodingthe FAM22 portion of the fusion protein. Thus, in some embodiments, afirst amplification primer is complementary or homologous to a portionof the nucleic acid that encodes the YWHAE portion of the fusion proteinand is complementary or homologous to a portion of the nucleic acid thatencodes the FAM22 portion of the fusion protein.

In some embodiments, the primer extension reaction and analysis isperformed using PYROSEQUENCING™ (Uppsala, Sweden) which essentially issequencing by synthesis. A sequencing primer is first hybridized to asingle stranded, PCR amplified DNA template from the individual, andincubated with the enzymes, DNA polymerase, ATP sulfurylase, luciferaseand apyrase, and the substrates, adenosine 5′ phosphosulfate (APS) andluciferin. One of four deoxynucleotide triphosphates (dNTP), forexample, corresponding to the nucleotide present in the mutation orpolymorphism, is then added to the reaction. DNA polymerase catalyzesthe incorporation of the dNTP into the standard DNA strand. Eachincorporation event is accompanied by release of pyrophosphate (PPi) ina quantity equimolar to the amount of incorporated nucleotide.Consequently, ATP sulfurylase converts PPi to ATP in the presence ofadenosine 5′ phosphosulfate. This ATP drives the luciferase-mediatedconversion of luciferin to oxyluciferin that generates visible light inamounts that are proportional to the amount of ATP. The light producedin the luciferase-catalyzed reaction is detected by a charge coupleddevice (CCD) camera and seen as a peak in a PYROGRAM™. Each light signalis proportional to the number of nucleotides incorporated and allows aclear determination of the presence or absence of, for example, themutation or polymorphism. Thereafter, apyrase, a nucleotide degradingenzyme, continuously degrades unincorporated dNTPs and excess ATP. Whendegradation is complete, another dNTP is added which corresponds to thedNTP present in for example the selected SNP. Addition of dNTPs isperformed one at a time. Deoxyadenosine alfa-thio triphosphate (dATPS)is used as a substitute for the natural deoxyadenosine triphosphate(dATP) since it is efficiently used by the DNA polymerase, but notrecognized by the luciferase. For detailed information about reactionconditions for the PYROSEQUENCING, see, e.g. U.S. Pat. No. 6,210,891,content of which is incorporated herein by reference in its entirety.

Alternatively, an INVADER® assay can be used (Third Wave Technologies,Inc (Madison, Wis.)). This assay is generally based upon astructure-specific nuclease activity of a variety of enzymes, which areused to cleave a target-dependent cleavage structure, thereby indicatingthe presence of specific nucleic acid sequences or specific variationsthereof in a sample (see, e.g. U.S. Pat. No. 6,458,535). For example, anINVADER® operating system (OS), provides a method for detecting andquantifying DNA and RNA. The INVADER® OS is based on a “perfect match”enzyme-substrate reaction. The INVADER® OS uses proprietary CLEAVASE®enzymes (Third Wave Technologies, Inc (Madison, Wis.)), which recognizeand cut only the specific structure formed during the INVADER® process.Unlike the PCR-based methods, the INVADER® OS relies on linearamplification of the signal generated by the INVADER® process, ratherthan on exponential amplification of the target.

In the INVADER® process, two short DNA probes hybridize to the target toform a structure recognized by the CLEAVASE® enzyme. The enzyme thencuts one of the probes to release a short DNA “flap.” Each released flapbinds to a fluorescently-labeled probe and forms another cleavagestructure. When the CLEAVASE® enzyme cuts the labeled probe, the probeemits a detectable fluorescence signal.

Another method to determine sequence of a nucleic acid is using “genechips”. The use of microarrays comprising a multiplicity of sequences isbecoming increasingly common in the art. Accordingly, a microarrayhaving at least one oligonucleotide probe, as described above, appendedthereon, can be used for interrogating the presence of a specificnucleic acid sequence in a sample. Probes can be affixed to surfaces foruse as “gene chips.” Such gene chips can be used to detect presence of anucleic acid in a sample by a number of techniques known to one of skillin the art. In one technique, oligonucleotides are arrayed on a genechip for determining the nucleotide sequence by the sequencing byhybridization approach, such as that outlined in U.S. Pat. Nos.6,025,136 and 6,018,041. The probes can also be used for fluorescentdetection of a genetic sequence. Such techniques have been described,for example, in U.S. Pat. Nos. 5,968,740 and 5,858,659. A probe also canbe affixed to an electrode surface for the electrochemical detection ofnucleic acid sequences such as described by Kayyem et al. U.S. Pat. No.5,952,172 and by Kelley, S. O. et al. (1999) Nucleic Acids Res.27:4830-4837.

In some embodiments, presence of the nucleic acid in the sample can bedone using Real-Time PCR. Real time PCR is an amplification techniquethat can be used to determine expression levels of mRNA corresponding toa protein of interest. (See, e.g., Gibson et al., Genome Research6:995-1001, 1996; Heid et al., Genome Research 6:986-994, 1996).Real-time PCR evaluates the level of PCR product accumulation duringamplification. This technique permits quantitative evaluation of mRNAlevels in multiple samples. For mRNA levels, mRNA can be extracted froma biological sample, e.g. a blood sample (such as white blood cellsand/or platelets) and cDNA is prepared using standard techniques.Real-time PCR can be performed, for example, using a PerkinElmer/Applied Biosystems (Foster City, Calif.) 7700 Prism instrument.Matching primers and fluorescent probes can be designed for genes ofinterest using, for example, the primer express program provided byPerkin Elmer/Applied Biosystems (Foster City, Calif.). Optimalconcentrations of primers and probes can be initially determined bythose of ordinary skill in the art, and control (for example,beta-actin) primers and probes can be obtained commercially from, forexample, Perkin Elmer/Applied Biosystems (Foster City, Calif.). Toquantitate the amount of the specific nucleic acid of interest in asample, a standard curve is generated using a control. Standard curvescan be generated using the Ct values determined in the real-time PCR,which are related to the initial concentration of the nucleic acid ofinterest used in the assay. Standard dilutions ranging from 10¹-10⁶copies of the gene of interest are generally sufficient. In addition, astandard curve is generated for the control sequence. This permitsstandardization of initial content of the nucleic acid of interest in atest sample to the amount of control for comparison purposes.

Methods of real-time quantitative PCR using TaqMan probes are well knownin the art. Detailed protocols for real-time quantitative PCR areprovided, for example, for RNA in: Gibson et al., 1996, A novel methodfor real time quantitative RT-PCR. Genome Res., 10:995-1001; and for DNAin: Heid et al., 1996, Real time quantitative PCR. Genome Res.,10:986-994.

The TaqMan based assays use a fluorogenic oligonucleotide probe thatcontains a 5′ fluorescent dye and a 3′ quenching agent. The probehybridizes to a PCR product, but cannot itself be extended due to ablocking agent at the 3′ end. When the PCR product is amplified insubsequent cycles, the 5′ nuclease activity of the polymerase, forexample, AmpliTaq, results in the cleavage of the TaqMan probe. Thiscleavage separates the 5′ fluorescent dye and the 3′ quenching agent,thereby resulting in an increase in fluorescence as a function ofamplification (see, for example, Perkin-Elmer).

In another embodiment, detection of RNA transcripts can be achieved byNorthern blotting, wherein a preparation of RNA is run on a denaturingagarose gel, and transferred to a suitable support, such as activatedcellulose, nitrocellulose or glass or nylon membranes. Labeled (e.g.,radiolabeled) cDNA or RNA is then hybridized to the preparation, washedand analyzed by methods such as autoradiography.

Detection of RNA transcripts can further be accomplished using knownamplification methods. For example, mRNA can be reverse-transcribed intocDNA followed by polymerase chain reaction (RT-PCR); or use a singleenzyme for both steps as described in U.S. Pat. No. 5,322,770, orreverse transcribe mRNA into cDNA followed by symmetric gap lipase chainreaction (RT-AGLCR) as described by R. L. Marshall, et al., PCR Methodsand Applications 4: 80-84 (1994). One suitable method for detectingenzyme mRNA transcripts is described in reference Pabic et. al.Hepatology, 37(5): 1056-1066, 2003, content of which is hereinincorporated by reference.

In situ hybridization visualization can also be employed, wherein aradioactively labeled antisense RNA probe is hybridized with nucleicacid encoding the fusion protein in a test sample, washed, cleaved withRNase and exposed to a sensitive emulsion for autoradiography. Thesamples can be stained with haematoxylin to demonstrate the histologicalcomposition of the sample, and dark field imaging with a suitable lightfilter shows the developed emulsion. Non-radioactive labels such asdigoxigenin can also be used.

Alternatively, mRNA expression can be detected on a DNA array, chip or amicroarray. Oligonucleotides corresponding to enzyme are immobilized ona chip which is then hybridized with labeled nucleic acids of a testsample obtained from a patient. Positive hybridization signal isobtained with the sample containing biomarker transcripts. Methods ofpreparing DNA arrays and their use are well known in the art. (See, forexample U.S. Pat. Nos: 6,618,6796; 6,379,897; 6,664,377; 6,451,536;548,257; U.S. 20030157485 and Schena et al. 1995 Science 20:467-470;Gerhold et al. 1999 Trends in Biochem. Sci. 24, 168-173; and Lennon etal. 2000 Drug discovery Today 5: 59-65, which are herein incorporated byreference). Serial Analysis of Gene Expression (SAGE) can also beperformed (See for example U.S. Patent Application 20030215858).

In some embodiments, the assay for detection of the nucleic acidcomprises a step of amplifying the nucleic acid before the detectionstep. This can be accomplished for example using a pair of primers thatamplify a portion of the nucleic acid comprising a portion that encodesat least a part of the YWHAE portion of the fusion protein and at leasta part of the FAM22 portion of the FAM22. For example, the pair ofprimers can be chosen as to amplify a region of the nucleic acidcomprising at least nucleotides 665, 666, 667, 668, 669, 670, 671, 672,673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686,687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700,701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714 or715 to 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728,729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742,743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756,757, 768, 769, 760, 761, 762, 763, 764, or 765 of SEQ ID NO: 1 or SEQ IDNO: 2.

In some embodiments, first primer in the primer pair is selected fromSEQ ID NO: 81 (5′-AGAGGCTGAGAGAGTC GGAGACA CTA-3′), SEQ ID NO: 82(5′-TATGGATGATCGAGAGGATCTGGTG-3′); and SEQ ID NO: 83 (5′-CAGAACTGGATACGC TGAGT GAAGAA-3′) and the second primer in the primer pair isSEQ ID NO: 84 (5′-CTCATAGACACT CCTGG GGTTACAGG-3′).

After amplification, the amplified nucleic acid can be subjected to DNAsequencing analysis. Exemplary DNA sequencing methods available to theskilled artisan and amenable to the assays, methods, systems andcompositions described herein are described elsewhere herein. In someembodiments, sequencing is using the BigDye Terminator Ready ReactionCycle Sequencing (Applied Biosystems).

By way of example only, presence of the fusion protein in the sample canbe determined by contacting the test sample with an antibody-basedbinding moiety that specifically binds to the fusion protein or to afragment thereof. Formation of the antibody-protein complex can then bedetected by a variety of methods known in the art.

As used herein, the term “antibody-based binding moiety” or “antibody”can include immunoglobulin molecules and immunologically activedeterminants of immunoglobulin molecules, e.g., molecules that containan antigen binding site which specifically binds to the fusion protein.The term “antibody-based binding moiety” is intended to include wholeantibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includesfragments thereof which are also specifically bind with the fusionprotein or a fragment thereof. Antibodies can be fragmented usingconventional techniques. Thus, the term includes segments ofproteolytically-cleaved or recombinantly-prepared portions of anantibody molecule that are capable of selectively reacting with acertain protein. Non-limiting examples of such proteolytic and/orrecombinant fragments include Fab, F(ab′)2, Fab′, Fv, dAbs and singlechain antibodies (scFv) containing a VL and VH domain joined by apeptide linker. The scFv's can be covalently or non-covalently linked toform antibodies having two or more binding sites. Thus, “antibody-basedbinding moiety” includes polyclonal, monoclonal, or other purifiedpreparations of antibodies and recombinant antibodies. The term“antibody-based binding moiety” is further intended to include humanizedantibodies, bispecific antibodies, and chimeric molecules having atleast one antigen binding determinant derived from an antibody molecule.In some embodiments, the antibody-based binding moiety can be detectablylabeled.

“Labeled antibody”, as used herein, includes antibodies that are labeledby a detectable means and include, but are not limited to, antibodiesthat are enzymatically, radioactively, fluorescently, andchemiluminescently labeled. Antibodies can also be labeled with adetectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, or HIS. Thedetection and quantification of the fusion protein in test samplescorrelate to the intensity of the signal emitted from the detectablylabeled antibody.

In some embodiments, the antibody-based binding moiety can be detectablylabeled by linking the antibody to an enzyme. The enzyme, in turn, whenexposed to its substrate, will react with the substrate in such a manneras to produce a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric or by visual means. Enzymes which canbe used to detectably label the antibodies against the fusion proteincan include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-V-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-VI-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

Detection can also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling an antibody, it ispossible to detect the antibody through the use of radioimmune assays.The radioactive isotope can be detected by such means as the use of agamma counter or a scintillation counter or by autoradiography. Isotopeswhich are particularly useful for the purpose of detection are ³H, ¹³¹I,³⁵S, ¹⁴C, and ¹²⁵I.

It is also possible to label an antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wavelength, its presence can then be detected due tofluorescence. Examples of the most commonly used fluorescent labelingcompounds include, but not limited to, CYE dyes, fluoresceinisothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine.

An antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

An antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-antibodyis then determined by detecting the presence of luminescence that arisesduring the course of a chemical reaction. Examples of chemiluminescentlabeling compounds can include, but not limited to, luminol, luciferin,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Without limitations, presence of fusion protein in the biological samplecan be detected by immunoassays, such as enzyme linked immunoabsorbantassay (ELISA), radioimmunoassay (RIA), Immunoradiometric assay (IRMA),Western blotting, immunocytochemistry or immunohistochemistry, each ofwhich are described in more detail below. In some embodiments,immunoassays such as ELISA or RIA can be used for determining presenceof the fusion protein in the sample. Antibody arrays or protein chipscan also be employed, see for example U.S. Patent Application Nos:2003/0013208A1; 2002/0155493A1; 2003/0017515 and U.S. Pat. Nos.6,329,209; 6,365,418, which are herein incorporated by reference.Commercially available antibodies and/or immunoassays (such as ELISA)for detecting YWHAE or FAM22 proteins, e.g., from Cell BioLabs, Abcam,Novus Biologicals, and Thermo Scientific Pierce Antibodies, can be usedin the assays and/or methods described herein.

The most common enzyme immunoassay is the “Enzyme-Linked ImmunosorbentAssay (ELISA).” ELISA is a technique for detecting and measuring theconcentration of an antigen using a labeled (e.g. enzyme linked) form ofthe antibody. There are different forms of ELISA, which are well knownto those skilled in the art. The standard techniques known in the artfor ELISA are described in “Methods in Immunodiagnosis”, 2nd Edition,Rose and Bigazzi, eds. John Wiley & Sons, 1980; Campbell et al.,“Methods and Immunology”, W. A. Benjamin, Inc., 1964; and Oellerich, M.1984, J. Clin. Chem. Clin. Biochem., 22:895-904.

In a “sandwich ELISA”, an antibody (e.g. anti-enzyme) is linked to asolid phase (i.e. a microtiter plate) and exposed to a biological samplecontaining antigen (e.g. enzyme). The solid phase is then washed toremove unbound antigen. A labeled antibody (e.g. enzyme linked) is thenbound to the bound-antigen (if present) forming anantibody-antigen-antibody sandwich. Examples of enzymes that can belinked to the antibody are alkaline phosphatase, horseradish peroxidase,luciferase, urease, and B-galactosidase. The enzyme linked antibodyreacts with a substrate to generate a colored reaction product that canbe measured.

In a “competitive ELISA”, antibody is incubated with a sample containingantigen (i.e. enzyme). The antigen-antibody mixture is then contactedwith a solid phase (e.g. a microtiter plate) that is coated with antigen(i.e., enzyme). The more antigen present in the sample, the less freeantibody that will be available to bind to the solid phase. A labeled(e.g., enzyme linked) secondary antibody is then added to the solidphase to determine the amount of primary antibody bound to the solidphase.

In an “immunohistochemistry assay” a test sample is tested for specificproteins by exposing the test sample to antibodies that are specific forthe protein that is being assayed. The antibodies are then visualized byany of a number of methods to determine the presence and amount of theprotein present. Examples of methods used to visualize antibodies are,for example, through enzymes linked to the antibodies (e.g., luciferase,alkaline phosphatase, horseradish peroxidase, or beta-galactosidase), orchemical methods (e.g., DAB/Substrate chromagen). The sample is thenanalysed microscopically, for example, by light microscopy of a samplestained with a stain that is detected in the visible spectrum, using anyof a variety of such staining methods and reagents known to thoseskilled in the art.

Alternatively, “Radioimmunoassays” can be employed. A radioimmunoassayis a technique for detecting and measuring the concentration of anantigen using a labeled (e.g. radioactively or fluorescently labeled)form of the antigen. Examples of radioactive labels for antigens include³H, ¹⁴C, and ¹²⁵I. The concentration of antigen enzyme in a test sampleor a biological sample can be measured by having the antigen in thebiological sample compete with the labeled (e.g. radioactively) antigenfor binding to an antibody to the antigen. To ensure competitive bindingbetween the labeled antigen and the unlabeled antigen, the labeledantigen is present in a concentration sufficient to saturate the bindingsites of the antibody. The higher the concentration of antigen in thesample, the lower the concentration of labeled antigen that will bind tothe antibody.

In a radioimmunoassay, to determine the concentration of labeled antigenbound to antibody, the antigen-antibody complex must be separated fromthe free antigen. One method for separating the antigen-antibody complexfrom the free antigen is by precipitating the antigen-antibody complexwith an anti-isotype antiserum. Another method for separating theantigen-antibody complex from the free antigen is by performing a“solid-phase radioimmunoassay” where the antibody is linked (e.g.,covalently) to Sepharose beads, polystyrene wells, polyvinylchloridewells, or microtiter wells. By comparing the concentration of labeledantigen bound to antibody to a standard curve based on samples having aknown concentration of antigen, the concentration of antigen in thebiological sample can be determined.

An “Immunoradiometric assay” (IRMA) is an immunoassay in which theantibody reagent is radioactively labeled. An IRMA requires theproduction of a multivalent antigen conjugate, by techniques such asconjugation to a protein e.g., rabbit serum albumin (RSA). Themultivalent antigen conjugate must have at least 2 antigen residues permolecule and the antigen residues must be of sufficient distance apartto allow binding by at least two antibodies to the antigen. For example,in an IRMA the multivalent antigen conjugate can be attached to a solidsurface such as a plastic sphere. Unlabeled “sample” antigen andantibody to antigen which is radioactively labeled are added to a testtube containing the multivalent antigen conjugate coated sphere. Theantigen in the sample competes with the multivalent antigen conjugatefor antigen antibody binding sites. After an appropriate incubationperiod, the unbound reactants are removed by washing and the amount ofradioactivity on the solid phase is determined. The amount of boundradioactive antibody is inversely proportional to the concentration ofantigen in the sample.

In some embodiments, Western blotting (Towbin et at., Proc. Nat. Acad.Sci. 76:4350 (1979)) can be used to presence of the fusion protein inthe sample, wherein a suitably treated sample is run on an SDS-PAGE gelbefore being transferred to a solid support, such as a nitrocellulosefilter. Detectably labeled anti-enzyme antibodies can then be used toassess enzyme levels, where the intensity of the signal from thedetectable label corresponds to the amount of enzyme present. Levels canbe quantified, for example by densitometry.

In addition to immunoassays, the presence of the fusion protein in thesample can also be determined by mass spectrometry such as MALDI/TOF(time-of-flight), SELDI/TOF, liquid chromatography-mass spectrometry(LC-MS), gas chromatography-mass spectrometry (GC-MS), high performanceliquid chromatography-mass spectrometry (HPLC-MS), capillaryelectrophoresis-mass spectrometry, nuclear magnetic resonancespectrometry, or tandem mass spectrometry (e.g., MS/MS, MS/MS/MS,ESI-MS/MS, etc.). See for example, U.S. Patent Application Nos:20030199001, 20030134304, 20030077616, content of all of which isincorporated herein by reference in their entirety. Mass spectrometrymethods are well known in the art and have been used to quantify and/oridentify molecules (see, e.g., Li et al. (2000) Tibtech 18:151-160;Rowley et al. (2000) Methods 20: 383-397; and Kuster and Mann (1998)Curr. Opin. Structural Biol. 8: 393-400).

In some embodiments, a gas phase ion spectrophotometer can be used. Inother embodiments, laser-desorption/ionization mass spectrometry can beused to analyze the sample. Modern laser desorption/ionization massspectrometry (“LDI-MS”) can be practiced in two main variations: matrixassisted laser desorption/ionization (“MALDI”) mass spectrometry andsurface-enhanced laser desorption/ionization (“SELDI”). In MALDI, theanalyte is mixed with a solution containing a matrix, and a drop of theliquid is placed on the surface of a substrate. The matrix solution thenco-crystallizes with the biological molecules. The substrate is insertedinto the mass spectrometer. Laser energy is directed to the substratesurface where it desorbs and ionizes the biological molecules withoutsignificantly fragmenting them. See, e.g., U.S. Pat. No. 5,118,937 andNo. 5,045,694, content of both of which is incorporated herein byreference in their entirety.

In SELDI, the substrate surface is modified so that it is an activeparticipant in the desorption process. In one variant, the surface isderivatized with adsorbent and/or capture reagents that selectively bindthe protein of interest. In another variant, the surface is derivatizedwith energy absorbing molecules that are not desorbed when struck withthe laser. In another variant, the surface is derivatized with moleculesthat bind the protein of interest and that contain a photolytic bondthat is broken upon application of the laser. In each of these methods,the derivatizing agent generally is localized to a specific location onthe substrate surface where the sample is applied. See, e.g., U.S. Pat.No. 5,719,060 and WO 98/59361, content of both of which is incorporatedherein by reference in their entirety. The two methods can be combinedby, for example, using a SELDI affinity surface to capture an analyteand adding matrix-containing liquid to the captured analyte to providethe energy absorbing material.

For additional information regarding mass spectrometers, see, e.g.,Principles of Instrumental Analysis, 3rd edition., Skoog, SaundersCollege Publishing, Philadelphia, 1985; and Kirk-Othmer Encyclopedia ofChemical Technology, 4^(th) ed. Vol. 15 (John Wiley & Sons, New York1995), pp. 1071-1094. Software programs such as the Biomarker Wizardprogram (Ciphergen Biosystems, Inc., Fremont, Calif.) can be used to aidin analyzing mass spectra, e.g., comparing the signal strength of peakvalues from spectra of a test subject sample and a control sample (e.g.,a normal healthy person). The mass spectrometers and their techniquesare well known to those of skill in the art.

In some embodiments, the assays, methods, and systems described hereincomprise contacting the biological sample with a reagent that binds withthe fusion protein or the nucleic acid. In some embodiments, thecontacting is under conditions that permit binding of the reagent to thefusion protein or the nucleic acid encoding the same.

In some embodiments, the reagent can be an antibody, or a fragmentthereof which retains binding to the fusion protein.

In some embodiments, the method further comprises contacting the samplewith a second reagent. The second reagent can be chosen so that it bindswith the fusion protein, the nucleic acid encoding the same, the firstreagent, a complex between the fusion protein and the first reagent, ora complex between the nucleic acid and the first reagent.

Without wishing to be bound by a theory, use of two or more differentreagents can be useful in “sandwich-type” detection assays. For example,the first regent can be used to capture an analyte (e.g., the fusionprotein or the nucleic acid) and the second reagent used for detectingthe first reagent—analyte complex.

Without limitation a reagent can be selected from the group consistingof nucleic acids, antibodies, antibody fragments, small molecules,polypeptides, peptides, peptidomimetics, lipids, saccharides, and thelike. In some embodiments, the reagent is an antibody, an antibodyfragment, or a nucleic acid. The reagent can be adapted to bind to thefusion protein, the nucleic acid encoding the same, or another reagent.The reagent can also be adapted for detecting the presence of the fusionprotein or the nucleic acid encoding the same in the sample.

In some embodiments, the reagent is a synthetic molecule or an isolatedmolecule.

In some embodiments, the reagent is an antibody that binds to an epitopedefined by amino acids 1-70 of the full length YWHAE. In someembodiments, the reagent is an antibody, or fragment thereof, that bindsto an epitope defined by amino acids 1-70 of SEQ ID NO: 3 or SEQ ID NO:4

In some embodiments, the reagent is an antibody that binds to an epitopedefined by amino acids 1-70 of the full length YWHAE. In someembodiments, the reagent is an antibody, or fragment thereof, that bindsto an epitope defined by amino acids 1-70 of SEQ ID NO: 3 or SEQ ID NO:4.

In some embodiments, the antibody is a rabbit antibody. In someembodiments, the antibody is antibody HPA008445 (Sigma).

In some embodiments, the reagent is an antibody that binds a portion ofSEQ ID No: 3 or SEQ ID NO: 4 but does not bind the full length YWHAE andthe FAM22 proteins. Methods for raising antibodies against a protein arewell known in the art and can be used to obtain antibodies that arespecific for the fusion-protein and do not bind to the full length YWHAEand the FAM22 proteins. For example, antibodies can be raised againstthe fusion protein and assayed for binding to the full length YWHAE orthe FAM22 proteins. Antibodies that do not bind to the full length YWHAEand the FAM22 proteins can be selected for the assays, methods, systems,kits, and compositions described herein.

In some embodiments, the detection method comprises: (i) contacting thebiological sample with: (a) a first reagent, e.g., antibody or fragmentthereof that binds with a full length YWHAE or FAM22 protein and thefusion protein; and (b) a second reagent, e.g., antibody or fragmentthat binds with the full length YWHAE or FAM22 protein but does not bindthe fusion protein; and (ii) detecting of binding of the first reagentand the second reagent in the sample, wherein binding of the firstreagent but not the second reagent indicating that a YWHAE-FAM22 fusionprotein is present in the sample. In some embodiments, the firstantibody can be antibody HPA008445 (Sigma) and the second antibody canbe BML-SA475R (Enzo Life Sciences).

In some embodiments, the reagent can be adapted to produce a signal whenbound to the fusion protein or the nucleic acid.

In some embodiments, the reagent comprises a label. As used herein, theterm “label” refers to any molecule that has a detectable property or iscapable of producing a detectable signal. The term “detectable property”means a physical or chemical property of a molecule that is capable ofindependent detection or monitoring by an analytical technique afterbeing conjugated with an affinity molecule, i.e., the property iscapable of being detected in the presence of a sample under analysis.The property can be light emission after excitation, quenching of aknown emission sites, electron spin, radio activity (electron emission,positron emission, alpha particle emission, etc.), nuclear spin, color,absorbance, near IR absorbance, UV absorbance, far UV absorbance, etc.As such, a label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means needed for the methods and devices described herein.

Suitable label moieties include fluorescent molecules, luminescentmolecules and nanoparticles, radioisotopes, chromophores, nucleotidechromophores, enzymes, substrates, chemiluminescent moieties,bioluminescent moieties, magnetic microbeads, magnetic nanoparticles,plasmonic nanoparticles, upconverting nanoparticles, bioluminescentmoieties, nanoparticles comprising fluorescent molecules, nanoparticlescomprising fluorophores, and the like. Means of detecting such labelsare well known to those of skill in the art. For example, radiolabelscan be detected using photographic film or scintillation counters,fluorescent markers can be detected using a photo-detector to detectemitted light. Enzymatic labels are typically detected by providing theenzyme with an enzyme substrate and detecting the reaction productproduced by the action of the enzyme on the enzyme substrate, andcalorimetric labels can be detected by visualizing the colored label. Assuch, a label moiety is any moiety detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Any method known in the art for detecting the particularlabel moiety can be used for detection.

The term “analytical technique” means an analytical chemical or physicalapproach or instrument for detecting and/or monitoring the property.Such instruments can be based on spectroscopic analytical methods suchas UV and visible light spectrometry, far IR, IR and near IRspectrometry, X-ray spectrometry, electron spin resonance spectrometry,nuclear magnetic resonance (NMR) spectrometry, etc.

In some embodiments, the label moiety is a luminescent nanoparticle.

In some embodiments, the label moiety can be a magnetic nanoparticle, aplasmonic nanoparticle, or an upconverting nanoparticle. As used herein,the term “plasmonic nanoparticle” refers to a nanoparticle that has verystrong absorption (and scattering) spectrum that is tunable by changingthe shape, the composition or the medium around their surfaces. It willbe appreciated that the term includes all plasmonic nanoparticles ofvarious shapes and surface surrounding which gives them surface plasmonabsorption and scattering spectrum in the visible-near infra-red regionof the spectrum. As used herein, an “upconverting nanoparticle” means ananoparticle which is a combination of an absorber which is excited byinfrared (IR) light and an emitter ion in a crystal lattice, whichconverts IR light into visible radiation.

In some embodiments, the label moiety is a fluorophore or fluorescentmolecule or dye. A wide variety of fluorescent molecules are known inthe art. Typically, the fluorophore is an aromatic or heteroaromaticcompound and can be a pyrene, anthracene, naphthalene, acridine,stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine,carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamineor other like compound. Exemplary fluorophores include, but are notlimited to, 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Carboxynapthofluorescein (pH 10); 5-Carboxytetramethylrhodamine(5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5-Hydroxy Tryptamine (HAT);5-ROX (carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine);6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin;7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin;9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA(9-Amino-6-chloro-2-methoxyacridine); Acridine Orange; Acridine Red;Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin(Photoprotein); Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™;Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™;Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™;Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S;AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin;Anilin Blue; Anthrocyl stearate; APC-Cy7; APTS; Astrazon Brilliant Red4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine;ATTO-TAGTm CBQCA; ATTO-TAGTm FQ; Auramine; Aurophosphine G;Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF(low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP(Y66H); BG-647; Bimane; Bisbenzamide; Blancophor FFG; Blancophor SV;BOBO™-1; BOBO™-3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570;Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X;Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6GSE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR;Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™-1; BO-PRO™-3; BrilliantSulphoflavin FF; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green;Calcium Green-1 Ca2+Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+;Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White;Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow;Catecholamine; CFDA; CFP-Cyan Fluorescent Protein; Chlorophyll;Chromomycin A; Chromomycin A; CMFDA; Coelenterazine; Coelenterazine cp;Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazinehcp; Coelenterazine ip; Coelenterazine O; Coumarin Phalloidin; CPMMethylcoumarin; CTC; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™;Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); d2; Dabcyl; Dansyl;Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansylfluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH(Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123);Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP); DIDS;Dihydorhodamine 123 (DHR); DiO (DiOC18(3)); DiR; DiR (DiIC18(7));Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97;Eosin; Erythrosin; Erythrosin ITC; Ethidium homodimer-1 (EthD-1);Euchrysin; Europium (III) chloride; Europium; EYFP; Fast Blue; FDA;Feulgen (Pararosaniline); FITC; FL-645; Flazo Orange; Fluo-3; Fluo-4;Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold(Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™(high pH); Fura-2, high calcium; Fura-2, low calcium; Genacryl BrilliantRed B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow5GF; GFP (S65T); GFP red shifted (rsGFP); GFP wild type, non-UVexcitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv;Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine(FluoroGold); Hydroxytryptamine; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1;LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS;Lissamine Rhodamine; Lissamine Rhodamine B; LOLO-1; LO-PRO-1; LuciferYellow; Mag Green; Magdala Red (Phloxin B); Magnesium Green; MagnesiumOrange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF;Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; MitotrackerGreen FM; Mitotracker Orange; Mitotracker Red; Mitramycin;Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS(Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red;Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow;Nylosan Brilliant Iavin E8G; Oregon Green™; Oregon Green 488-X; OregonGreen™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue;Pararosaniline (Feulgen); PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5;PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; PhorwiteBKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26; PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline;Procion Yellow; Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin;RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200; Rhodamine B extra;Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine;Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal;R-phycoerythrin (PE); red shifted GFP (rsGFP, S65T); S65A; S65C; S65L;S65T; Sapphire GFP; Serotonin; Sevron Brilliant Red 2B; Sevron BrilliantRed 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™;sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (super glow GFP); SITS; SITS(Primuline); SITS (Stilbene Isothiosulphonic Acid); SPQ(6-methoxy-N-(3-sulfopropyl)-quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine G Extra; Tetracycline; Tetramethylrhodamine;Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); ThiazineRed R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN;Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR;TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC(TetramethylRodaminelsoThioCyanate); True Blue; TruRed; Ultralite;Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1;and YOYO-3. Many suitable forms of these fluorescent molecules areavailable and can be used.

Other exemplary label moieties include radiolabels (e.g., ³H, ¹²⁵I, ³⁵S,¹⁴C, or ³²P), enzymes (e.g., galactosidases, glucorinidases,phosphatases (e g, alkaline phosphatase), peroxidases (e.g., horseradishperoxidase), and cholinesterases), and calorimetric labels such ascolloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, and latex) beads. Patents teaching the use of such labelmoieties include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350,3,996,345, 4,277,437, 4,275,149, and 4,366,241, content of each of whichis incorporated herein by reference in its entirety.

In some embodiments, the label is a fluorescent, luminescent, orradioactive label.

In some embodiments, the reagent can be immobilized to support. Forexample, the reagent can be covalently or non-covalently linked to asolid support or the reagent can be immobilized within a matrixmaterial. Methods for immobilizing a reagent to solid support are wellknown in the art and available to one of skill in the art.

After a subject is identified as needing anti-cancer treatment, thesubject can be treated with an anti-cancer treatment. Thus, in anotheraspect provided herein is method of treating cancer in a subject in needthereof, the method comprising administering an anti-cancer therapy ortreatment to the subject, wherein the subject expresses a YWHAE-FAM22fusion protein or a nucleic acid encoding the same. In some embodiments,the method comprising: assaying a biological sample from a subject forpresence of the YWHAE-FAM22 fusion protein or a nucleic acid encodingthe same and administering an anti-cancer therapy or treatment to thesubject if the YWHAE-FAM22 fusion protein or a nucleic acid encoding thesame is detected in the sample.

As used herein, an anti-cancer treatment aims to reduce, prevent oreliminate cancer cells or the spread of cancer cells or the symptoms ofcancer in the local, regional or systemic circulation. Anti-cancertreatment also means the direct treatment of tumors, for example byreducing or stabilizing their number or their size (curative effect),but also by preventing the in situ progression of tumor cells or theirdiffusion, or the establishment of tumors; this also includes thetreatment of deleterious effects linked to the presence of such tumors,in particular the attenuation of symptoms observed in a patient or animprovement in quality of life. By “reduced” in the context of cancer ismeant reduction of at least 10% in the growth rate of a tumor or thesize of a tumor or cancer cell burden. Exemplary anti-cancer therapiesinclude, but are not limited to, radiation, drug, surgery to removetumors, and the like. Many anti-cancer therapies are known to one ofskill in the art and can be used with the treatment method describedherein.

In some embodiments, the anti-cancer therapy comprises administering aneffective amount of an anti-cancer agent to the subject.

As used herein, the term “anti-cancer agent” is refers to any compound(including its analogs, derivatives, prodrugs and pharmaceuticallysalts) or composition which can be used to treat cancer. Anti-cancercompounds for use in the present invention include, but are not limitedto, inhibitors of topoisomerase I and II, alkylating agents, microtubuleinhibitors (e.g., taxol), and angiogenesis inhibitors. Exemplaryanti-cancer compounds include, but are not limited to, paclitaxel(taxol); docetaxel; germicitibine; Aldesleukin; Alemtuzumab;alitretinoin; allopurinol; altretamine; amifostine; anastrozole; arsenictrioxide; Asparaginase; BCG Live; bexarotene capsules; bexarotene gel;bleomycin; busulfan intravenous; busulfanoral; calusterone;capecitabine; carboplatin; carmustine; carmustine with PolifeprosanImplant; celecoxib; chlorambucil; cisplatin; cladribine;cyclophosphamide; cytarabine; cytarabine liposomal; dacarbazine;dactinomycin; actinomycin D; Darbepoetin alfa; daunorubicin liposomal;daunorubicin, daunomycin; Denileukin diftitox, dexrazoxane; docetaxel;doxorubicin; doxorubicin liposomal; Dromostanolone propionate; Elliott'sB Solution; epirubicin; Epoetin alfa estramustine; etoposide phosphate;etoposide (VP-16); exemestane; Filgrastim; floxuridine (intraarterial);fludarabine; fluorouracil (5-FU); fulvestrant; gemtuzumab ozogamicin;goserelin acetate; hydroxyurea; Ibritumomab Tiuxetan; idarubicin;ifosfamide; imatinib mesylate; Interferon alfa-2a; Interferon alfa-2b;irinotecan; letrozole; leucovorin; levamisole; lomustine (CCNU);mechlorethamine (nitrogenmustard); megestrol acetate; melphalan (L-PAM);mercaptopurine (6-MP); mesna; methotrexate; methoxsalen; mitomycin C;mitotane; mitoxantrone; nandrolone phenpropionate; Nofetumomab; LOddC;Oprelvekin; oxaliplatin; pamidronate; pegademase; Pegaspargase;Pegfilgrastim; pentostatin; pipobroman; plicamycin; mithramycin;porfimer sodium; procarbazine; quinacrine; Rasburicase; Rituximab;Sargramostim; streptozocin; talbuvidine (LDT); talc; tamoxifen;temozolomide; teniposide (VM-26); testolactone; thioguanine (6-TG);thiotepa; topotecan; toremifene; Tositumomab; Trastuzumab; tretinoin(ATRA); Uracil Mustard; valrubicin; valtorcitabine (monoval LDC);vinblastine; vinorelbine; zoledronate; and any mixtures thereof. In someembodiments, the anti-cancer agent is a paclitaxel-carbohydrateconjugate, e.g., a paclitaxel-glucose conjugate, as described in U.S.Pat. No. 6,218,367, content of which is herein incorporated by referencein its entirety.

In yet another aspect provided herein is an assay for selecting atreatment regime for a subject with cancer. The assay comprising:detecting the presence of YWHAE-FAM22 fusion protein or a nucleic acidencoding the same in a biological sample taken from the subject; and ifat least one of the fusion protein or the nucleic acid is detected, thenselecting, and optionally administering, a treatment regimen comprisingan anti-cancer therapy to the subject.

Also provided herein is an isolated sample from a subject, wherein thesample comprises a YWHAE-FAM22 fusion protein or a nucleic acid encodingthe same. In some embodiments, the sample further comprises a firstreagent that can bind with the fusion protein or the nucleic acid.

The invention also provides a composition comprising: (i) a YWHAE-FAM22fusion protein or a nucleic acid encoding the YWHAE-FAM22 fusionprotein, wherein the YWHAE-FAM22 fusion protein or the nucleic acid isat least partially isolated from a biological sample obtained from asubject; and (ii) a reagent that binds with the fusion protein or thenucleic acid. In some embodiments, the YWHAE-FAM22 fusion protein or thenucleic acid is in a biological sample obtained from a subject.

Reagents that can bind with the fusion protein or the nucleic acids aredescribed in detail herein. In some embodiments, the reagent is adaptedto produce a signal so as to detect presence of the fusion protein orthe nucleic acid in the sample.

In another aspect provided herein is an isolated fusion protein,comprising a portion of a YWHAE protein and a portion of a FAM-22protein. In some embodiments, the fusion protein consists of amino acidsequence of SEQ ID NO: 3 or SEQ ID NO: 4.

Peptide modifications are well known in the art. Thus, a fusion proteindescribed herein can comprise one or more peptide modifications known inthe art. Exemplary peptide modifications for modifying the fusionprotein described herein include, but are not limited to, D amino acids,α amino acids, β amino acids, non-amide or modified amide linkages,chemically modified amino acids, naturally occurring non-proteogenicamino acids, rare amino acids, chemically synthesized compounds thathave properties known in the art to be characteristic of an amino acid,and the like.

An isolated nucleic acid encoding the fusion protein is also providedherein. In some embodiments, the nucleic acid comprises a 5′-hydroxylgroup and/or a 3′-hydroxyl group. In some embodiments, the nucleic acidencoding the fusion protein consists of the nucleotide sequence of SEQID NO: 1 or SEQ ID NO: 2.

Modified nucleic acids are well known in the art. Thus, a nucleic aciddescribed herein can comprise one or more nucleic acid modificationsknown in the art. For example, In the nucleic acid can comprise one ormore nucleic acid modifications selected from the group consisting ofinternucleotide linkage modifications (intersugar linkagemodifications), sugar modifications, nucleobase modifications, backbonemodifications/replacements, and any combinations thereof. Exemplaryinternucleotide linkage modifications include, but are not limited to,phosphorothioate, phosphorodithioate, phosphotriester (e.g. alkylphosphotriester), aminoalkylphosphotriester, alkyl-phosphonate (e.g.,methyl-phosphonate), selenophosphate, phosphoramidate (e.g.,N-alkylphosphoramidate), boranophosphonate, and the like. Exemplarysugar modifications include, but are not limited to, 2′-O-Me(2′-O-methyl), 2′-O-MOE (2′-O-methoxyethyl), 2′-F,2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), 2′-S-methyl,2′-O—CH₂-(4′-C) (LNA), 2′-O—CH₂CH₂-(4′-C) (ENA), 2′-O-aminopropyl(2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), arabinose sugar, and the like. Exemplary nucleobasemodifications include, but are not limited to, inosine, xanthine,hypoxanthine, nubularine, isoguanisine, tubercidine, 5-methylcytosine(5-me-C); 5-hydroxymethyl cytosine; xanthine; hypoxanthine;2-aminoadenine; 6-methyl and other 6-alkyl derivatives of adenine andguanine; 2-propyl and other 2-alkyl derivatives of adenine and guanine;2-thiouracil; 2-thiothymine; 2-thiocytosine; 5-propynyl uracil;5-propynyl cytosine; 6-azouracil; 6-azocytosine; 6-azothymine; 5-uracil(pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines; 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines; 7-methyl and other 7-alkyl derivatives of adenine andguanine; 8-azaguanine; 8-azaadenine; 7-deazaguanine; 7-deazaadenine;3-deazaguanine; 3-deazaadenin; universal base; and any combinationsthereof. Exemplary backbone modifications include, but are not limitedto, morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA),aminoethylglycyl PNA (aegPNA), backnone-extended pyrrolidine PNA(bepPNA), and the like.

In another aspect provided herein are systems (and computer readablemedia for causing computer systems) for use in the assays and methodsdescribed herein. For example for use in an assay or method foridentifying a subject suitable for cancer treatment based on presence ofthe fusion protein or the nucleic acid encoding the same in a biologicalsample taken from the subject.

Generally the system comprises: (i) a determination module configured toreceive at least one test sample (e.g., biological sample) and performat least one analysis on the at least one test sample to determinepresence of the fusion protein or a nucleic acid encoding the same; (ii)a storage device configured to store output data from the determinationmodule; (iii) a computing module, e.g., a non-human machine, comprisingspecifically-programmed instructions to determine from the output datathe presence of the fusion protein or the nucleic acid encoding thesame; and (iv) a display module for displaying a content based in parton the data output from the computing module, wherein the contentcomprises a signal indicative of the presence of the fusion protein orthe nucleic acid.

In some embodiments, the system comprises a biological sample taken froma subject and a reagent that binds with the YWHAE-FAM22 fusion proteinor a nucleic acid encoding the same.

In some embodiments, the determination module can be configured toperform DNA sequencing to determine the presence of the nucleic acidencoding the fusion protein.

In some embodiments, the determination module can be configured tocapture the fusion protein or the nucleic acid encoding the same.

In some embodiments, the determination module can further comprise acomparison module adapted to compare the data output from thedetermination module with reference data stored on the storage device.In some embodiments, the reference data can include, but not limited to,at least a part of the amino acid sequence for the fusion protein, atleast a part of the nucleic acid sequence for the nucleic acid encodingthe fusion protein, length of the fusion protein or the nucleic acid,molecular weight of the fusion protein or the nucleic acid, and anycombinations thereof.

In some embodiments, the display module can also comprise instructionsfor displaying a content based in part on the data output from thecomparison module. In some embodiments, the content displayed on thedisplay module can further comprise a signal indicative of the subjectrecommended to receive a treatment regimen for ESS.

In some embodiments, the storage device of the computer system can befurther configured to store information of at least one subject to betested. Examples of the information can include, but is not limited to,medical history, family history, physical parameter, gender, and thelike.

A tangible and non-transitory (e.g., not transitory forms of signaltransmission) computer readable medium having computer readableinstructions recorded thereon to define software modules forimplementing a method on a computer is also provided herein. In oneembodiment, the computer readable storage medium comprises: (i)instructions for comparing the data stored on a storage device withreference data to provide a comparison result, wherein the comparisonidentifies the presence or absence of the fusion protein or the nucleicacid encoding the same; and (ii) instructions for displaying a contentbased in part on the data output from the determination module, whereinthe content comprises a signal indicative of the presence of the fusionprotein or the nucleic acid, and optionally the absence of the fusionprotein or the nucleic acid. In some embodiments, the content cancomprise a signal indicative of the subject needing treatment for ESS.

In some embodiments, the instructions can be specifically programmed toperform a comparison to identify the presence of a nucleic acidcomprising a nucleotide sequence homologous to the nucleic acid sequenceof the nucleic acid encoding the fusion protein.

In some embodiments, the instructions can be specifically programmed toperform an analysis of binding of a reagent to the fusion protein or thenucleic acid encoding the same.

In some embodiments, the computer readable medium can further compriseinstructions to identify the presence or absence of the fusion proteinor the nucleic acid encoding the same.

The computer readable media can be any available tangible media that canbe accessed by a computer. Computer readable media includes volatile andnonvolatile, removable and non-removable tangible media implemented inany method or technology for storage of information such as computerreadable instructions, data structures, program modules or other data.Computer readable media includes, but is not limited to, RAM (randomaccess memory), ROM (read only memory), EPROM (eraseable programmableread only memory), EEPROM (electrically eraseable programmable read onlymemory), flash memory or other memory technology, CD-ROM (compact discread only memory), DVDs (digital versatile disks) or other opticalstorage media, magnetic cassettes, magnetic tape, magnetic disk storageor other magnetic storage media, other types of volatile andnon-volatile memory, and any other tangible medium which can be used tostore the desired information and which can accessed by a computerincluding and any suitable combination of the foregoing.

In some embodiments, the computer readable storage media 700 can includethe “cloud” system, in which a user can store data on a remote server,and later access the data or perform further analysis of the data fromthe remote server.

An embodiment of the computer system can be illustrated with referenceto FIGS. 9 and 10. Computer-readable data embodied on one or morecomputer-readable media, or computer readable medium 1000, can defineinstructions, for example, as part of one or more programs, that, as aresult of being executed by a computer, instruct the computer to performone or more of the functions described herein (e.g., in relation tosystem 900, or computer readable medium 1000), and/or variousembodiments, variations and combinations thereof. Such instructions canbe written in any of a plurality of programming languages, for example,Java, J#, Visual Basic, C, C#, C++, Fortran, Pascal, Eiffel, Basic,COBOL assembly language, and the like, or any of a variety ofcombinations thereof. The computer-readable media on which suchinstructions are embodied can reside on one or more of the components ofeither of system 900, or computer readable medium 1000 described herein,may be distributed across one or more of such components, and may be intransition there between.

The computer-readable media can be transportable such that theinstructions stored thereon can be loaded onto any computer resource toimplement the assays and/or methods described herein. In addition, itshould be appreciated that the instructions stored on the computerreadable media, or computer-readable medium 1000, described above, arenot limited to instructions embodied as part of an application programrunning on a host computer. Rather, the instructions may be embodied asany type of computer code (e.g., software or microcode) that can beemployed to program a computer to implement the assays and/or methodsdescribed herein. The computer executable instructions may be written ina suitable computer language or combination of several languages. Basiccomputational biology methods are known to those of ordinary skill inthe art and are described in, for example, Setubal and Meidanis et al.,Introduction to Computational Biology Methods (PWS Publishing Company,Boston, 1997); Salzberg, Searles, Kasif, (Ed.), Computational Methods inMolecular Biology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler,Bioinformatics Basics: Application in Biological Science and Medicine(CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: APractical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc.,2nd ed., 2001).

The functional modules of certain embodiments of the system describedherein can include a determination module, a storage device, and adisplay module. In some embodiments, the system can further include acomparison module. The functional modules can be executed on one, ormultiple, computers, or by using one, or multiple, computer networks.The determination module 902 has computer executable instructions toprovide sequence information in computer readable form. As used herein,“sequence information” refers to any nucleotide and/or amino acidsequence, including but not limited to full-length nucleotide and/oramino acid sequences, partial nucleotide and/or amino acid sequences, ormutated sequences. Moreover, information “related to” the sequenceinformation includes detection of the presence or absence of a sequence(e.g., detection of a mutation or deletion), determination of theconcentration of a sequence in the sample (e.g., amino acid sequenceexpression levels, or nucleotide (RNA or DNA) expression levels), andthe like. The term “sequence information” is intended to include thepresence or absence of post-translational modifications (e.g.phosphorylation, glycosylation, summylation, farnesylation, and thelike).

As an example, determination modules 902 for determining sequenceinformation may include known systems for automated sequence analysisincluding but not limited to Hitachi FMBIO® and Hitachi FMBIO® IIFluorescent Scanners (available from Hitachi Genetic Systems, Alameda,Calif.); Spectrumedix® SCE 9610 Fully Automated 96-CapillaryElectrophoresis Genetic Analysis Systems (available from SpectruMedixLLC, State College, Pa.); ABI PRISM® 377 DNA Sequencer, ABI® 373 DNASequencer, ABI PRISM® 310 Genetic Analyzer, ABI PRISM® 3100 GeneticAnalyzer, and ABI PRISM® 3700 DNA Analyzer (available from AppliedBiosystems, Foster City, Calif.); Molecular Dynamics FluorImager™ 575,SI Fluorescent Scanners, and Molecular Dynamics FluorImager™ 595Fluorescent Scanners (available from Amersham Biosciences UK Limited,Little Chalfont, Buckinghamshire, England); GenomyxSC™ DNA SequencingSystem (available from Genomyx Corporation (Foster City, Calif.); andPharmacia ALF™ DNA Sequencer and Pharmacia ALFexpress™ (available fromAmersham Biosciences UK Limited, Little Chalfont, Buckinghamshire,England).

Alternative methods for determining sequence information, i.e.determination modules 902, include systems for protein and DNA analysis.For example, mass spectrometry systems including Matrix Assisted LaserDesorption Ionization—Time of Flight (MALDI-TOF) systems andSELDI-TOF-MS ProteinChip array profiling systems; systems for analyzinggene expression data (see, for example, published U.S. PatentApplication, Pub. No. U.S. 2003/0194711); systems for array basedexpression analysis: e.g., HT array systems and cartridge array systemssuch as GeneChip® AutoLoader, Complete GeneChip® Instrument System,GeneChip® Fluidics Station 450, GeneChip® Hybridization Oven 645,GeneChip® QC Toolbox Software Kit, GeneChip® Scanner 3000 7G plusTargeted Genotyping System, GeneChip® Scanner 3000 7G Whole-GenomeAssociation System, GeneTitan™ Instrument, and GeneChip® Array Station(each available from Affymetrix, Santa Clara, Calif.); automated ELISAsystems (e.g., DSX® or DS2® (available from Dynax, Chantilly, Va.) orthe Triturus® (available from Grifols USA, Los Angeles, Calif.), TheMago® Plus (available from Diamedix Corporation, Miami, Fla.);Densitometers (e.g. X-Rite-508-Spectro Densitometer® (available from RPImaging™, Tucson, Ariz.), The HYRYS™ 2 HIT densitometer (available fromSebia Electrophoresis, Norcross, Ga.); automated Fluorescence insituhybridization systems (see for example, U.S. Pat. No. 6,136,540); 2D gelimaging systems coupled with 2-D imaging software; microplate readers;Fluorescence activated cell sorters (FACS) (e.g. Flow CytometerFACSVantage SE, (available from Becton Dickinson, Franklin Lakes, N.J.);and radio isotope analyzers (e.g. scintillation counters).

The sequence information and/or expression level information determinedin the determination module can be read by the storage device 904. Asused herein the “storage device” 904 is intended to include any suitablecomputing or processing apparatus or other device configured or adaptedfor storing data or information. Examples of electronic apparatussuitable for use with the system described herein can includestand-alone computing apparatus, data telecommunications networks,including local area networks (LAN), wide area networks (WAN), Internet,Intranet, and Extranet, and local and distributed computer processingsystems. Storage devices 604 also include, but are not limited to:magnetic storage media, such as floppy discs, hard disc storage media,magnetic tape, optical storage media such as CD-ROM, DVD, electronicstorage media such as RAM, ROM, EPROM, EEPROM and the like, general harddisks and hybrids of these categories such as magnetic/optical storagemedia. The storage device 604 is adapted or configured for havingrecorded thereon sequence information or expression level information.Such information may be provided in digital form that can be transmittedand read electronically, e.g., via the Internet, on diskette, via USB(universal serial bus) or via any other suitable mode of communication,e.g., the “cloud”.

As used herein, “expression level information” refers to any nucleotideand/or amino acid expression level information, including but notlimited to full-length nucleotide and/or amino acid sequences, partialnucleotide and/or amino acid sequences, or mutated sequences. Moreover,information “related to” the expression level information includesdetection of the presence or absence of a sequence (e.g., presence orabsence of an amino acid sequence, nucleotide sequence, or posttranslational modification), determination of the concentration of asequence in the sample (e.g., amino acid sequence levels, or nucleotide(RNA or DNA) expression levels, or level of post translationalmodification), and the like. In some embodiments, the expression levelinformation also includes arithmetic manipulation of expression levels.

As used herein, “stored” refers to a process for encoding information onthe storage device 904. Those skilled in the art can readily adopt anyof the presently known methods for recording information on known mediato generate manufactures comprising the sequence information orexpression level information.

A variety of software programs and formats can be used to store thesequence information or expression level information on the storagedevice. Any number of data processor structuring formats (e.g., textfile or database) can be employed to obtain or create a medium havingrecorded thereon the sequence information or expression levelinformation.

By providing sequence information and/or expression level information incomputer-readable form, one can use the sequence information and/orexpression level information in readable form in the comparison module906 to compare a specific sequence or expression profile with thereference data within the storage device 904. For example, searchprograms can be used to identify fragments or regions of the sequencesthat match a particular sequence (reference data, e.g., sequenceinformation of major or rare alleles corresponding to the SNPs describedherein) or direct comparison of the determined expression level can becompared to the reference data expression level (e.g., median expressionlevel information obtained from a population of subjects). Thecomparison made in computer-readable form provides a computer readablecomparison result which can be processed by a variety of means. Content908 based on the comparison result can be retrieved from thedetermination module 902 or the comparison module 906 to indicate thepresence or absence of the fusion protein or the nucleic acid encodingthe same.

In one embodiment the reference data stored in the storage device 904 tobe read by the determination module 902 or the comparison module 906 issequence information data obtained from a control biological sample ofthe same type as the biological sample to be tested. Alternatively, thereference data are a database, e.g., a part of the entire genomesequence of an organism, or a protein family of sequences, or anexpression level profile (RNA, protein or peptide). In one embodiment,the reference data are sequence information and/or expression levelprofiles that are used to facilitate determining whether a subjectshould be recommended for a treatment regimen for ESS.

In some embodiments, the reference data are one or more referencepolynucleotide, or polypeptide sequences. In some embodiments, thereference polynucleotide sequences can be derived from nucleotidesequences selected SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments,the reference polypeptide sequences can be derived from amino acidsequences of SEQ ID NO: 3 or SEQ ID NO: 4.

In one embodiment, the reference data are electronically or digitallyrecorded and annotated from databases including, but not limited toGenBank (NCBI) protein and DNA databases such as genome, ESTs, SNPS,Traces, Celara, Ventor Reads, Watson reads, HGTS, and the like; SwissInstitute of Bioinformatics databases, such as ENZYME, PROSITE,SWISS-2DPAGE, Swiss-Prot and TrEMBL databases; the Melanie softwarepackage or the ExPASy WWW server, and the like; the SWISS-MODEL,Swiss-Shop and other network-based computational tools; theComprehensive Microbial Resource database (available from The Instituteof Genomic Research). The resulting information can be stored in arelational data base that may be employed to determine homologiesbetween the reference data or genes or proteins within and amonggenomes.

The “comparison module” 906 can use a variety of available softwareprograms and formats for the comparison operative to compare sequenceinformation determined in the determination module 902 to referencedata. In one embodiment, the comparison module 906 is configured to usepattern recognition techniques to compare sequence information from oneor more entries to one or more reference data patterns. The comparisonmodule 906 can be configured using existing commercially-available orfreely-available software for comparing patterns, and may be optimizedfor particular data comparisons that are conducted. The comparisonmodule 906 provides computer readable information related to thesequence information that can include, for example, detection of thepresence or absence of a sequence (e.g., detection of a mutation ordeletion (protein or DNA), information regarding distinct alleles,detection of post-translational modification, or omission or repetitionof sequences); determination of the concentration of a sequence in thesample (e.g., amino acid sequence/protein expression levels, ornucleotide (RNA or DNA) expression levels, or levels ofpost-translational modification), or determination of an expressionprofile.

In one embodiment, the comparison module 906 permits the prediction ofprotein sequences from polynucleotide sequences, permits prediction ofopen reading frames (ORF), or permits prediction of homologous sequenceinformation in comparison to reference data, i.e., homologous proteindomains, homologous DNA or RNA sequences, or homologous exons and/orintrons.

In one embodiment, the comparison module 906 uses sequence informationalignment programs such as BLAST (Basic Local Alignment Search Tool) orFAST (using the Smith-Waterman algorithm) may be employed individuallyor in combination. These algorithms determine the alignment betweensimilar regions of sequences and a percent identity between sequences.For example, alignment may be calculated by matching, bases-by-base oramino acid-by amino-acid.

The comparison module 906, or any other module of the system describedherein, can include an operating system (e.g., UNIX) on which runs arelational database management system, a World Wide Web application, anda World Wide Web server. World Wide Web application includes theexecutable code necessary for generation of database language statements(e.g., Structured Query Language (SQL) statements). Generally, theexecutables will include embedded SQL statements. In addition, the WorldWide Web application may include a configuration file which containspointers and addresses to the various software entities that comprisethe server as well as the various external and internal databases whichmust be accessed to service user requests. The Configuration file alsodirects requests for server resources to the appropriate hardware—as maybe necessary should the server be distributed over two or more separatecomputers. In one embodiment, the World Wide Web server supports aTCP/IP protocol. Local networks such as this are sometimes referred toas “Intranets.” An advantage of such Intranets is that they allow easycommunication with public domain databases residing on the World WideWeb (e.g., the GenBank or Swiss Pro World Wide Web site). Thus, in aparticular embodiment, users can directly access data (via Hypertextlinks for example) residing on Internet databases using a HTML interfaceprovided by Web browsers and Web servers. In another embodiment, userscan directly access data residing on the “cloud” provided by the cloudcomputing service providers.

In one embodiment, the comparison module 906 performs comparisons withmass-spectrometry spectra, for example comparisons of peptide fragmentsequence information can be carried out using spectra processed inMATLAB with script called “Qcealign” (see for example WO2007/022248,herein incorporated by reference) and “Qpeaks” (Spectrum SquareAssociates, Ithaca, N.Y.), or Ciphergen Peaks 2.1™ software. Theprocessed spectra can then be aligned using alignment algorithms thatalign sample data to the control data using minimum entropy algorithm bytaking baseline corrected data (see for example WIPO PublicationWO2007/022248, herein incorporated by reference). The comparison resultcan be further processed by calculating ratios. Protein expressionprofiles can be discerned.

In one embodiment, computational algorithms are used in the comparisonmodule 906 such as expectation-maximization (EM), subtraction and PHASEare used in methods for statistical estimation of haplotypes (see, e.g.,Clark, A. G. Mol Biol Evol 7:111-22 (1990); Stephens, M., Smith, N. J. &Donnelly, P. Am J Hum Genet 68:978-89 (2001); Templeton, A. R., Sing, C.F., Kessling, A. & Humphries, Genetics 120:1145-54 (1988)).

Various algorithms are available which are useful for comparing data andidentifying the predictive gene signatures. For example, algorithms suchas those identified in Xu et al., Physiol. Genomics 11:11-20 (2002).There are numerous software available for detection of SNPs andpolymorphisms that can be used in the comparison module, including, butnot limited to: HaploSNPer, a web-based program for detecting SNPs andalleles in user-specified input sequences from both diploid andpolyploid species (available on the world-wide web atbioinformatics.nl/tools/haplosnper/; see also Tang et al., BMC Genetics9:23 (2008)); Polybayes, a tool for SNP discovery in redundant DNAsequences (March, G T., et al., Nature Genetics 23(4):452-6 (1999);SSAHA-SNP, a polymorphism detection tool that uses the SSAHA alignmentalgorithm (available from Wellcome Trust Sanger Institute, Cambridge,United Kingdom, see also Ning Z., et al., Genome Research 11(10):1725-9(2001)); Polyphred, A SNP discovery package built on phred, phrap, andconsed tools (available on the world-wide web, see Nickerson, D A etal., Nucleic Acids Research 25(14):2745-51 (1997)); NovoSNP, a graphicalJava-based program (PC/Mac/Linux) to identify SNPs and indels (availableon the world-wide web, see Weckx, S. et al., Genome Research15(3):436-442 (2005)); SNPdetector™, for automated identification ofSNPs and mutations in fluorescence-based resequencing reads (availablefrom Affymetrix, Santa Clara, Calif.), see also Thang et al. PLoS ComputBiol (5):e53 (2005). SNPdetector runs on Unix/Linux platform and isavailable publicly; Affymetrix (Santa Clara, Calif.) has multiple dataanalysis software that can be used, for example Genotyping Console™Software, GeneChip® Sequence Analysis Software (GSEQ), GeneChip®Targeted Genotyping Analysis Software (GTGS) and Expression Console™Software.

In one embodiment, the comparison module 906 compares gene expressionprofiles. For example, detection of gene expression profiles can bedetermined using Affymetrix Microarray Suite software version 5.0 (MAS5.0) (available from Affymetrix, Santa Clara, Calif.) to analyze therelative abundance of a gene or genes on the basis of the intensity ofthe signal from probe sets, and the MAS 5.0 data files can betransferred into a database and analyzed with Microsoft Excel andGeneSpring 6.0 software (available from Agilent Technologies, SantaClara, Calif.). The detection algorithm of MAS 5.0 software can be usedto obtain a comprehensive overview of how many transcripts are detectedin given samples and allow a comparative analysis of two or moremicroarray data sets.

In one embodiment, the comparison module 906 compares protein expressionprofiles. Any available comparison software can be used, including butnot limited to, the Ciphergen Express (CE) and Biomarker PatternsSoftware (BPS) package (available from Ciphergen Biosystems, Inc.,Freemont, Calif.). Comparative analysis can be done with protein chipsystem software (e.g., The Proteinchip Suite (available from Bio-RadLaboratories, Hercules, Calif.). Algorithms for identifying expressionprofiles can include the use of optimization algorithms such as the meanvariance algorithm (e.g. JMP Genomics algorithm available from JMPSoftware Cary, N.C.).

In one embodiment, pattern comparison software can be used to determinewhether patterns of expression or mutations are indicative of thepresence or the absence of the conditions detected in a test sample of asubject.

The comparison module 906 provides computer readable comparison resultthat can be processed in computer readable form by predefined criteria,or criteria defined by a user, to provide content based in part on thecomparison result that may be stored and output as requested by a userusing a display module 910. The display module 910 enables display of acontent 908 based in part on the comparison result for the user, whereinthe content 908 is a signal indicative of the presence or absence of thefusion protein or the nucleic acid encoding the same. Such signal canbe, for example, a display of content 908 can be on a computer monitor,a printed page, or a light or sound indicative of indicative of thepresence or absence of the fusion protein or the nucleic acid encodingthe same.

In various embodiments of the computer system described herein, thecomparison module 906 can be integrated into the determination module902.

The content 908 based on the comparison result can also include anexpression profile of the fusion protein. In one embodiment, the content908 based on the comparison includes a sequence of a particular gene orprotein. In one embodiment, the content 908 based on the comparisonresult is merely a signal indicative of the presence or absence of thefusion protein or the nucleic acid encoding the same. In someembodiments, the content 908 can be a signal indicative of the subjectrecommended to receive a treatment regimen for treating ESS.

In one embodiment, the content 908 based on the comparison result isdisplayed a on a computer monitor. In one embodiment, the content 908based on the comparison result is displayed through printable media. Thedisplay module 910 can be any suitable device configured to receive froma computer and display computer readable information to a user.Non-limiting examples include, for example, general-purpose computerssuch as those based on Intel PENTIUM-type processor, Motorola PowerPC,Sun UltraSPARC, Hewlett-Packard PA-RISC processors, any of a variety ofprocessors available from Advanced Micro Devices (AMD) of Sunnyvale,Calif., or any other type of processor, visual display devices such asflat panel displays, cathode ray tubes and the like, as well as computerprinters of various types.

In one embodiment, a World Wide Web browser is used for providing a userinterface for display of the content 908 based on the comparison result.It should be understood that other modules of the system describedherein can be adapted to have a web browser interface. Through the Webbrowser, a user may construct requests for retrieving data from thecomparison module. Thus, the user will typically point and click to userinterface elements such as buttons, pull down menus, scroll bars and thelike conventionally employed in graphical user interfaces. The requestsso formulated with the user's Web browser are transmitted to a Webapplication which formats them to produce a query that can be employedto extract the pertinent information related to the sequenceinformation, e.g., display of an indication of the presence or absenceof mutation or deletion (DNA or protein); display of expression levelsof an amino acid sequence (protein); display of nucleotide (RNA or DNA)expression levels; display of expression, SNP, or mutation profiles, orhaplotypes, or display of information based thereon. In one embodiment,the sequence information of the reference sample data is also displayed.

In any embodiments, the comparison module can be executed by computerimplemented software as discussed earlier. In such embodiments, a resultfrom the comparison module can be displayed on an electronic display.The result can be displayed by graphs, numbers, characters or words. Inadditional embodiments, the results from the comparison module can betransmitted from one location to at least one other location. Forexample, the comparison results can be transmitted via any electronicmedia, e.g., internet, fax, phone, a “cloud” system, and anycombinations thereof. Using the “cloud” system, users can store andaccess personal files and data or perform further analysis on a remoteserver rather than physically carrying around a storage medium such as aDVD or thumb drive.

The system 900 and computer readable medium 1000 are merely illustrativeembodiments for performing assays for identifying a subject fortreatment for ESS, based on presence of the fusion protein or thenucleic acid encoding the same in sample taken from the subject, and isnot intended to limit the scope of the inventions described herein.Variations of system 900 and computer readable medium 1000 are possibleand are intended to fall within the scope of the inventions describedherein.

The modules of the machine, or used in the computer readable medium, canassume numerous configurations. For example, function can be provided ona single machine or distributed over multiple machines.

Kits

In yet another aspect provided herein are kits for use in the assay,methods, systems and compositions described herein. Accordingly,provided herein include kits for identifying a subject for endometrialstromal sarcoma or assessing the endometrial stromal sarcoma status in asubject. The kits can include at least one reagent adapted for detectingfor the presence or absence of the fusion protein or the nucleic acidencoding the same. The kits can also include instructions fordetermining that the subject is recommended for a treatment regimen fortreatment of ESS.

In some embodiments, the kit can comprise a solid substrate supportaffixed with at least one reagent that can bind (e.g., specificallybind) to the fusion protein or the nucleic acid encoding the same.Exemplary solid substrate support can include, but not limited to, amicrotiter plate for ELISA, a dipstick, a magnetic bead, or anycombinations thereof. Different solid substrate supports can be selectedbased on various types of assays, e.g., but not limited to, Westernblot, enzyme linked absorbance assay, mass spectrometry, immunoassay,flow cytometry, immunohistochemical analysis, and any combinationsthereof.

In some embodiments, the kit comprises at least one reagent adapted fordetecting presence of a YWHAE-FAM22 fusion protein or a nucleic acidencoding the same.

In some embodiments, the kit comprises at least one reagent thatspecifically binds the fusion protein or the nucleic acid.

In some embodiments, the kit comprises a first reagent and a secondreagent, wherein each can bind to the fusion protein or the nucleicacid. In some embodiments, the first reagent binds the YWHAE portion ofthe fusion protein or the nucleic acid encoding the same and the secondreagent binds the FAM-22 portion of the fusion protein or the nucleicacid encoding the same. By way of an example only, the first reagent canbe an antibody that binds with a YWHAE protein and the second reagentcan be an antibody that binds with a FAM-22 protein. In another example,the first and second reagents can be nucleic acid primers, wherein thefirst reagent (i.e., primer) is complementary or homologous to a portionof the nucleic acid encoding the YWHAE portion of the fusion protein anda second reagent (i.e., primer) is complementary or homologous to aportion of the nucleic acid encoding the FAM-22 portion of the fusionprotein

In some embodiments, the kit can comprise an oligonucleotide arrayaffixed with a plurality of oligonucleotide probes that interrogate asample for the presence of a nucleic acid encoding the fusion protein,and an optional container containing a detectable label (e.g.,comprising a fluorescent molecule) to be conjugated to a nucleotidemolecule derived from the sample.

Examples of reagents additional reagents that can be included the kitcan include, but are not limited to, buffers, reagents for detection,and the like.

In some embodiments, the kit comprises a fusion protein or a nucleicacid encoding the same.

Some Selected Definitions

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected herein. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below. Unless explicitly stated otherwise,or apparent from context, the terms and phrases below do not exclude themeaning that the term or phrase has acquired in the art to which itpertains. The definitions are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention,because the scope of the invention is limited only by the claims.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used to described the present invention,in connection with percentages means±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%,±4%, ±4.5%, or ±5%. The term “about” when used in connection withpercentages may mean±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%, ±4%, ±4.5%, or±5% of the value being referred to.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

As used herein the terms “comprising” or “comprises” means “including”or “includes” and are used in reference to compositions, methods,systems, and respective component(s) thereof, that are useful to theinvention, yet open to the inclusion of unspecified elements, whetheruseful or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, systems, andrespective components thereof as described herein, which are exclusiveof any element not recited in that description of the embodiment.

The abbreviation, “e.g.” is derived from the Latin exempli gratia, andis used herein to indicate a non-limiting example. Thus, theabbreviation “e.g.” is synonymous with the term “for example.”

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means at least two standarddeviation (2SD) away from a reference level. The term refers tostatistical evidence that there is a difference. It is defined as theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true.

The term “derivative” as used herein refers to a chemical substancerelated structurally to another, i.e., an “original” substance, whichcan be referred to as a “parent” compound. A “derivative” can be madefrom the structurally-related parent compound in one or more steps. Insome embodiments, the general physical and chemical properties of aderivative can be similar to or different from the parent compound.

The term “nucleic acid” is well known in the art. A “nucleic acid” asused herein will generally refer to a molecule (i.e., strand) of DNA,RNA or a derivative or analog thereof, comprising a nucleobase. Anucleobase includes, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g. an adenine “A,” a guanine “G” athymine “T” or a cytosine “C”) or RNA (e.g. an A, a G. an uracil “U” ora C). The term “nucleic acid” encompasses the terms “oligonucleotide”and “polynucleotide,” each as a subgenus of the term “nucleic acid.” Theterm “oligonucleotide” refers to a molecule of between about 3 and about100 nucleobases in length. The term “polynucleotide” refers to at leastone molecule of greater than about 100 nucleobases in length.

The term “nucleic acid sequence” refers to a single or double-strandedpolymer of deoxyribonucleotide or ribonucleotide bases read from the 5′to the 3′-end. It includes chromosomal DNA, self-replicating plasmids,infectious polymers of DNA or RNA and DNA or RNA that performs aprimarily structural role. “Nucleic acid sequence” also refers to aconsecutive list of abbreviations, letters, characters or words, whichrepresent nucleotides. In one embodiment, a nucleic acid can be a“probe” which is a relatively short nucleic acid, usually less than 100nucleotides in length.

The term “oligonucleotide,” as used herein refers to primers and probesdescribed herein, and is defined as a nucleic acid molecule comprised ofat least two or more ribo- or deoxyribonucleotides. The exact size ofthe oligonucleotide will depend on various factors and on the particularapplication and use of the oligonucleotide. The term “probe” as usedherein refers to an oligonucleotide, polynucleotide or nucleic acid,either RNA or DNA, whether occurring naturally as in a purifiedrestriction enzyme digest or produced synthetically, which is capable ofannealing with or specifically hybridizing to a nucleic acid withsequences complementary to the probe. A probe can be eithersingle-stranded or double-stranded. The exact length of the probe willdepend upon many factors, including temperature, source of probe and themethod used. For example, for diagnostic applications, depending on thecomplexity of the target sequence, an oligonucleotide probe typicallycontains 15-25 or more nucleotides, although it may contain fewernucleotides. The probes as disclosed herein are selected to besubstantially complementary to different strands of a particular targetnucleic acid sequence. This means that the probes must be sufficientlycomplementary so as to be able to “specifically hybridize” or annealwith their respective target strands. Therefore, the probe sequence neednot reflect the exact complementary sequence of the target. For example,a non-complementary nucleotide fragment may be attached to the 5′ or 3′end of the probe, with the remainder of the probe sequence beingcomplementary to the target strand. Alternatively, non-complementarybases or longer sequences can be interspersed into the probe, providedthat the probe sequence has sufficient complementarily with the sequenceof the target nucleic acid to anneal therewith specifically.

In the context of some embodiments of various aspects described herein,the term “probe” refers to a molecule which can detectably distinguishbetween target molecules differing in structure (e.g. nucleic acid orprotein sequence). Detection can be accomplished in a variety ofdifferent ways depending on the type of probe used and the type oftarget molecule. Thus, for example, detection may be based ondiscrimination on detection of specific binding. Examples of suchspecific binding include antibody binding and nucleic acid, antibodybinding to protein, nucleic acid binding to nucleic acid, or aptamerbinding to protein or nucleic acid. Thus, for example, probes caninclude enzyme substrates, antibodies and antibody fragments, andpreferably nucleic acid hybridization probes.

The term “specifically hybridize” refers to the association between twosingle-stranded nucleic acid molecules of sufficient complementarysequence to permit such hybridization under pre-determined conditionsgenerally used in the art (sometimes the sequences are referred to as“substantially complementary”). In particular, the term specificallyhybridize also refers to hybridization of an oligonucleotide with asubstantially complementary sequence as compared to non-complementarysequence.

The term “specifically” as used herein with reference to a probe whichis used to specifically detect a sequence difference, refers to a probethat identifies a particular sequence difference based on exclusivehybridization to the sequence difference under stringent hybridizationconditions and/or on exclusive amplification or replication of thesequence difference.

In its broadest sense, the term “substantially” as used herein inrespect to “substantially complementary”, or when used herein withrespect to a nucleotide sequence in relation to a reference or targetnucleotide sequence, means a nucleotide sequence having a percentage ofidentity between the substantially complementary nucleotide sequence andthe exact complementary sequence of the reference or target nucleotidesequence of at least 60%, at least 70%, at least 80% or 85%, at least90%, at least 93%, at least 95% or 96%, at least 97% or 98%, at least99% or 100% (the later being equivalent to the term “identical” in thiscontext). For example, identity is assessed over a length of at least 10nucleotides, or at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22or up to 50 nucleotides of the entire length of the nucleic acidsequence to the reference sequence (if not specified otherwise below).Sequence comparisons can be carried out using default GAP analysis withthe University of Wisconsin GCG, SEQWEB application of GAP, based on thealgorithm of Needleman and Wunsch (Needleman and Wunsch (1970) J MoI.Biol. 48: 443-453; as defined above). A nucleotide sequence“substantially complementary” to a reference nucleotide sequencehybridizes to the reference nucleotide sequence under low stringencyconditions, preferably medium stringency conditions, most preferablyhigh stringency conditions.

In its broadest sense, the term “substantially identical”, when usedherein with respect to a nucleotide sequence, means a nucleotidesequence corresponding to a reference or target nucleotide sequence,wherein the percentage of identity between the substantially identicalnucleotide sequence and the reference or target nucleotide sequence isat least 60%, at least 70%, at least 80% or 85%, at least 90%, at least93%, at least 95% or 96%, at least 97% or 98%, at least 99% or 100% (thelater being equivalent to the term “identical” in this context). Forexample, identity is assessed over a length of 10-22 nucleotides, suchas at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or up to50 nucleotides of a nucleic acid sequence to the reference sequence (ifnot specified otherwise below). Sequence comparisons are carried outusing default GAP analysis with the University of Wisconsin GCG, SEQWEBapplication of GAP, based on the algorithm of Needleman and Wunsch(Needleman and Wunsch (1970) J MoI. Biol. 48: 443-453; as definedabove). A nucleotide sequence “substantially identical” to a referencenucleotide sequence hybridizes to the exact complementary sequence ofthe reference nucleotide sequence (i.e. its corresponding strand in adouble-stranded molecule) under low stringency conditions, preferablymedium stringency conditions, most preferably high stringency conditions(as defined above). Homologues of a specific nucleotide sequence includenucleotide sequences that encode an amino acid sequence that is at least24% identical, at least 35% identical, at least 50% identical, at least65% identical to the reference amino acid sequence, as measured usingthe parameters described above, wherein the amino acid sequence encodedby the homolog has the same biological activity as the protein encodedby the specific nucleotide. The term “substantially non-identical”refers to a nucleotide sequence that does not hybridize to the nucleicacid sequence under stringent conditions. The term “substantiallyidentical”, when used herein with respect to a polypeptide, means aprotein corresponding to a reference polypeptide, wherein thepolypeptide has substantially the same structure and function as thereference protein, e.g. where only changes in amino acids sequence notaffecting the polypeptide function occur. When used for a polypeptide oran amino acid sequence, the percentage of identity between thesubstantially similar and the reference polypeptide or amino acidsequence is at least 24%, at least 30%, at least 45%, at least 60%, atleast 75%, at least 90%, at least 95%, at least 99%, using default GAPanalysis parameters as described above. Homologues are amino acidsequences that are at least 24% identical, more preferably at least 35%identical, yet more preferably at least 50% identical, yet morepreferably at least 65% identical to the reference polypeptide or aminoacid sequence, as measured using the parameters described above, whereinthe amino acid sequence encoded by the homolog has the same biologicalactivity as the reference polypeptide.

The term “primer” as used herein refers to an oligonucleotide, eitherRNA or DNA, either single-stranded or double-stranded, either derivedfrom a biological system, generated by restriction enzyme digestion, orproduced synthetically which, when placed in the proper environment, isable to functionally act as an initiator of template-dependent nucleicacid synthesis. When presented with an appropriate nucleic acidtemplate, suitable nucleoside triphosphate precursors of nucleic acids,a polymerase enzyme, suitable cofactors and conditions such as asuitable temperature and pH, the primer may be extended at its 3′terminus by the addition of nucleotides by the action of a polymerase orsimilar activity to yield a primer extension product. The primer mayvary in length depending on the particular conditions and requirement ofthe application. For example, in diagnostic applications, theoligonucleotide primer is typically 15-25 or more nucleotides in length.The primer must be of sufficient complementarity to the desired templateto prime the synthesis of the desired extension product, that is, to beable to anneal with the desired template strand in a manner sufficientto provide the 3′ hydroxyl moiety of the primer in appropriatejuxtaposition for use in the initiation of synthesis by a polymerase orsimilar enzyme. It is not required that the primer sequence represent anexact complement of the desired template. For example, anon-complementary nucleotide sequence may be attached to the 5′ end ofan otherwise complementary primer. Alternatively, non-complementarybases may be interspersed within the oligonucleotide primer sequence,provided that the primer sequence has sufficient complementarity withthe sequence of the desired template strand to functionally provide atemplate-primer complex for the synthesis of the extension product.

The term “complementary” or “complement” as used herein refers to thebroad concept of sequence complementarity between regions of two nucleicacid strands or between two regions of the same nucleic acid strand. Itis known that an adenine residue of a first nucleic acid region iscapable of forming specific hydrogen bonds (“base pairing”) with aresidue of a second nucleic acid region which is anti-parallel to thefirst region if the residue is thymine or uracil. Similarly, it is knownthat a cytosine residue of a first nucleic acid strand is capable ofbase pairing with a residue of a second nucleic acid strand which isanti-parallel to the first strand if the residue is guanine. A firstregion of a nucleic acid is complementary to a second region of the sameor a different nucleic acid if, when the two regions are arranged in ananti-parallel fashion, at least one nucleotide residue of the firstregion is capable of base pairing with a residue of the second region.Preferably, the first region comprises a first portion and the secondregion comprises a second portion, whereby, when the first and secondportions are arranged in an anti-parallel fashion, such that at leastabout 50%, and preferably at least about 75%, at least about 90%, or atleast about 95% or at least 100% of the nucleotide residues of the firstportion are capable of base pairing with nucleotide residues in thesecond portion. More preferably, all nucleotide residues of the firstportion are capable of base pairing with nucleotide residues in thesecond portion.

As used herein, the term “epitope” means that portion of protein that isrecognized by a particular antibody. As such, the term “epitope”designates a specific amino acid sequence, modified amino acid sequence,or protein secondary or tertiary structure which is recognized by anantibody.

As used herein, the term “anti-cancer activity” or “anti-cancerproperties” refers to the inhibition (in part or in whole) or preventionof unregulated cell growth and/or the inhibition (in part or in whole)or prevention of a cancer as defined herein. Anticancer activityincludes, e.g., the ability to reduce, prevent, or repair geneticdamage, modulate undesired cell proliferation, modulate misregulatedcell death, or modulate mechanisms of metastasis (e.g., ability tomigrate).

By “treatment”, “prevention” or “amelioration” of a disease or disorderis meant delaying or preventing the onset of such a disease or disorder,reversing, alleviating, ameliorating, inhibiting, slowing down orstopping the progression, aggravation or deterioration the progressionor severity of a condition associated with such a disease or disorder.In some embodiments, one or more symptoms of a disease or disorder arealleviated by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, or at least 50%.

As used herein, the term “cancer” refers to an uncontrolled growth ofcells that may interfere with the normal functioning of the bodilyorgans and systems. Cancers that migrate from their original locationand seed vital organs can eventually lead to the death of the subjectthrough the functional deterioration of the affected organs. Ametastasis a cancer cell or group of cancer cells, distinct from theprimary tumor location resulting from the dissemination of cancer cellsfrom the primary tumor to other parts of the body. At the time ofdiagnosis of the primary tumor mass, the subject may be monitored forthe presence of in transit metastases, e.g., cancer cells in the processof dissemination. As used herein, the term cancer, includes, but is notlimited to the following types of cancer, breast cancer, biliary tractcancer, bladder cancer, brain cancer including Glioblastomas andmedulloblastomas; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer, gastric cancer; hematologicalneoplasms including acute lymphocytic and myelogenous leukemia; T-cellacute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronicmyelogenous leukemia, multiple myeloma; AIDS-associated leukemias andadult T-cell leukemia lymphoma; intraepithelial neoplasms includingBowen's disease and Paget's disease; liver cancer; lung cancer;lymphomas including Hodgkin's disease and lymphocytic lymphomas;neuroblastomas; oral cancer including squamous cell carcinoma; ovariancancer including those arising from epithelial cells, stromal cells,germ cells and mesenchymal cells; pancreatic cancer; prostate cancer;rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma,liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer includingmelanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma,and squamous cell cancer; testicular cancer including germinal tumorssuch as seminoma, non-seminoma (teratomas, choriocarcinomas), stromaltumors, and germ cell tumors; thyroid cancer including thyroidadenocarcinoma and medullar carcinoma; and renal cancer includingadenocarcinoma, Wilms tumor. Examples of cancer include but are notlimited to, carcinoma, including adenocarcinoma, lymphoma, blastoma,melanoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, small-cell lung cancer, non-smallcell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin'slymphoma, pancreatic cancer, Glioblastoma, cervical cancer, ovariancancer, liver cancer such as hepatic carcinoma and hepatoma, bladdercancer, breast cancer, colon cancer, colorectal cancer, endometrialcarcinoma, salivary gland carcinoma, kidney cancer such as renal cellcarcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostatecancer, vulval cancer, thyroid cancer, testicular cancer, esophagealcancer, and various types of head and neck cancer. Other cancers will beknown to the artisan.

In some embodiments, cancer is an endometrial sarcoma. Withoutlimitations, the endometrial cancer can be any subtypes, for example,serous, mucinous, and endometrioid histological subtypes.

As used herein, the term “precancerous condition” has its ordinarymeaning, i.e., an unregulated growth without metastasis, and includesvarious forms of hyperplasia and benign hypertrophy. Accordingly, a“precancerous condition” is a disease, syndrome, or finding that, ifleft untreated, can lead to cancer. It is a generalized state associatedwith a significantly increased risk of cancer. Premalignant lesion is amorphologically altered tissue in which cancer is more likely to occurthan its apparently normal counterpart. Examples of pre-malignantconditions include, but are not limited to, oral leukoplakia, actinickeratosis (solar keratosis), Barrett's esophagus, atrophic gastritis,benign hyperplasia of the prostate, precancerous polyps of the colon orrectum, gastric epithelial dysplasia, adenomatous dysplasia, hereditarynonpolyposis colon cancer syndrome (HNPCC), Barrett's esophagus, bladderdysplasia, precancerous cervical conditions, and cervical dysplasia.

As used herein, a “subject” means a human or animal. Examples ofsubjects include primates (e.g., humans, and monkeys). Usually theanimal is a vertebrate such as a primate, rodent, domestic animal orgame animal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments of the aspects describedherein, the subject is a mammal, e.g., a primate, e.g., a human. Theterms, “patient” and “subject” are used interchangeably herein. Theterms, “patient” and “subject” are used interchangeably herein. Asubject can be male or female. A subject can be one who has not beenpreviously diagnosed with cancer, e.g. endometrial stromal sarcoma.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of cancer.In addition, the methods and compositions described herein can be usedto treat domesticated animals and/or pets.

A subject can be one who has been previously diagnosed with cancer.Without wishing to be bound by a theory, the assays, methods, systems,kits and compositions described herein can be used to diagnose and/orclassify endometrial cancer in the subject. The assays, methods,systems, kits and compositions described herein can further compriseselecting a subject who has cancer. The method can also comprise thestep of diagnosing a subject for cancer before onset of administrationor treatment regime.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition which is effective forproducing some desired therapeutic effect in at least a sub-populationof cells in an animal at a reasonable benefit/risk ratio applicable toany medical treatment.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeuticallyeffective amount can vary with the subject's history, age, condition,sex, as well as the severity and type of the medical condition in thesubject, and administration of other pharmaceutically active agents.

The therapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the therapeutic which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Levels in plasmacanbe measured, for example, by high performance liquid chromatography.The effects of any particular dosage can be monitored by a suitablebioassay.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage canvary within this range depending upon the dosage form employedand the route of administration utilized.

The dosage can be determined by a physician and adjusted, as necessary,to suit observed effects of the treatment. Generally, the anti-canceragents are administered at a dose from 1 μg/kg to 150 mg/kg, 1 μg/kg to100 mg/kg, 1 μg/kg to 50 mg/kg, 1 μg/kg to 20 mg/kg, 1 μg/kg to 10mg/kg, 1 μg/kg to 1 mg/kg, 100 μg/kg to 100 mg/kg, 100 μg/kg to 50mg/kg, 100 μg/kg to 20 mg/kg, 100 μg/kg to 10 mg/kg, 100 μg/kg to 1mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 20 mg/kg, 1mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, or 10mg/kg to 20 mg/kg. It is to be understood that ranges given here includeall intermediate ranges, for example, the range 1 mg/kg to 10 mg/kgincludes 1 mg/kg to 2 mg/kg, 1 mg/kg to 3 mg/kg, 1 mg/kg to 4 mg/kg, 1mg/kg to 5 mg/kg, 1 mg/kg to 6 mg/kg, 1 mg/kg to 7 mg/kg, 1 mg/kg to 8mg/kg, 1 mg/kg to 9 mg/kg, 2 mg/kg to 10 mg/kg, 3 mg/kg to 10 mg/kg, 4mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 6 mg/kg to 10 mg/kg, 7 mg/kg to10 mg/kg, 8 mg/kg to 10 mg/kg, 9 mg/kg to 10 mg/kg, and the like. It isto be further understood that the ranges intermediate to the given aboveare also within the scope of this invention, for example, in the range 1mg/kg to 10 mg/kg, dose ranges such as 2 mg/kg to 8 mg/kg, 3 mg/kg to 7mg/kg, 4 mg/kg to 6 mg/kg, and the like.

With respect to duration and frequency of treatment, it is typical forskilled clinicians to monitor subjects in order to determine when thetreatment is providing therapeutic benefit, and to determine whether toincrease or decrease dosage, increase or decrease administrationfrequency, discontinue treatment, resume treatment or make otheralteration to treatment regimen. The dosing schedule can vary from oncea week to daily depending on a number of clinical factors, such as thesubject's sensitivity to the conjugates described herein. The desireddose can be administered everyday or every third, fourth, fifth, orsixth day. The desired dose can be administered at one time or dividedinto subdoses, e.g., 2-4 subdoses and administered over a period oftime, e.g., at appropriate intervals through the day or otherappropriate schedule. Such sub-doses can be administered as unit dosageforms. In some embodiments of the aspects described herein,administration is chronic, e.g., one or more doses daily over a periodof weeks or months. Examples of dosing schedules are administrationdaily, twice daily, three times daily or four or more times daily over aperiod oft week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months,4 months, 5 months, or 6 months or more.

As used herein, the term “administer” refers to the placement of acomposition into a subject by a method or route which results in atleast partial localization of the composition at a desired site suchthat desired effect is produced. Routes of administration suitable forthe methods of the invention include both local and systemicadministration. Generally, local administration results in more of thecomposition being delivered to a specific location as compared to theentire body of the subject, whereas, systemic administration results indelivery to essentially the entire body of the subject.

An anti-cancer agent can be administered by any appropriate route knownin the art including, but not limited to, oral or parenteral routes,including intravenous, intramuscular, subcutaneous, transdermal, airway(aerosol), pulmonary, nasal, rectal, vaginal, and topical (including onthe skin, and body cavities, such as buccal, vaginal, rectal andsublingual) administration.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and infrasternal injection and infusion. In someembodiments of the aspects described herein, the compositions areadministered by intravenous infusion or injection. In some embodiments,administration is oral.

As used herein, the term “isolated” or “purified” means that thematerial in question has been removed from its host, and associatedimpurities reduced or eliminated. Essentially, it means an objectspecies is the predominant species present (i.e., on a molar basis it ismore abundant than any other individual species in the composition), andpreferably a substantially purified fraction is a composition whereinthe object species comprises at least about 30 percent (on a molarbasis) of all other species present. Generally, a substantially purecomposition will comprise more than about 80 to 90 percent of allspecies present in the composition. Most preferably, the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.With reference to nucleic acid molecules, the term “isolated nucleicacid” refers to a nucleic acid molecule that is manipulated or modifiedby hand-of-man to remove at least one component with which it isgenerally found in its natural environment. Thus, the term “isolatednucleic acid” refers to a nucleic acid sequence that is separated fromsequences with which it is immediately contiguous (in the 5′ and 3′directions) in the naturally occurring genome of the organism from whichit was derived. With reference to proteins, the term “isolated protein”refers to a protein that is manipulated or modified by hand-of-man toremove at least one component with which it is generally found in itsnatural environment. Generally, isolating a compound from its naturalenvironment entails at least some manipulation of the target componentsuch that the isolated target component can be said to have beenmanipulated by hand-of-man and thus in some aspect different from as itoccurred in nature. In other words, an isolated compound can beconsidered as a compound that is does not occur in nature.

Embodiments of the various aspects described herein can also bedescribed by any one of the following paragraphs.

-   1. A method of identifying a subject suitable for endometrial    stromal sarcoma (ESS) treatment, the method comprising a step of    detecting in a biological sample taken from the subject presenting a    symptom of ESS the presence of a YWHAE-FAM22 fusion protein or a    nucleic acid encoding the same, wherein detection of the fusion    protein or the nucleic acid in the biological sample indicates that    the individual should undergo ESS treatment.-   2. The method of paragraph 1, wherein the nucleic acid comprises the    nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.-   3. The method of any of paragraphs 1 or 2, wherein the fusion    protein comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID    NO: 4.-   4. The method of any of paragraphs 1-3, wherein the sample is    selected from the group consisting of blood, urine, plasma, tissue,    cell, and any combinations thereof.-   5. The method of any of paragraphs 1-4, wherein the subject is a    mammal.-   6. The method of any of paragraphs 1-5, wherein the subject is    human.-   7. The method of any of paragraphs 1-6, wherein method comprises    contacting the sample with a first reagent that binds with the    fusion gene or the fusion protein.-   8. The method of paragraph 7, wherein the first reagent is selected    from the group consisting of a nucleic acid, an antibody, a small    molecule, a polypeptide, a peptide, a lipid, and any combinations    thereof.-   9. The method of paragraph 7 or 8, wherein the first reagent further    comprises a label to produce a signal so as to detect presence of    the fusion gene or the fusion protein in the sample.-   10. The method of paragraph 9, wherein the label is selected from    the group consisting of a radiolabel, a chromophore, a fluorophore,    an enzyme, and any combinations thereof.-   11. The method of any of paragraphs 7-10, wherein the first reagent    is covalently or non-covalently linked to a solid support.-   12. The method of paragraph 11, wherein the solid support is    selected from the group consisting of a chip, a microarray, a gel, a    test strip, and any combinations thereof.-   13. The method of any of paragraphs 7-12, wherein the sample    comprises a second reagent, wherein the second reagent binds with    the first reagent, the fusion gene, or the fusion protein.-   14. The method of paragraph 13, wherein the second reagent is    selected from the group consisting of a nucleic acid, an antibody, a    small molecule, a polypeptide, a peptide, a lipid, and any    combinations thereof.-   15. The method of paragraph 13 or 14, wherein the second reagent    further comprises a label to produce a signal so as to detect    presence of the first reagent bound to the fusion gene or the fusion    protein in the isolated sample.-   16. The method of paragraph 15, wherein the label is selected from    the group consisting of fluorophores, enzymes, and any combinations    thereof.-   17. The method of any of paragraphs 13-16, wherein the second    reagent is covalently or non-covalently linked to a solid support.-   18. The method of paragraph 17, wherein the solid support is    selected from the group consisting of a chip, a microarray, a gel, a    test strip, and any combinations thereof.-   19. An assay for selecting a treatment regimen for a subject with    endometrial stromal sarcoma, the assay comprising subjecting a    biological sample from the subject to:    -   (i) at least one protein detection assay adapted to determine        the presence of a YWHAE-FAM22 fusion protein; or    -   (ii) at least one nucleic acid sequence detection assay adapted        to determine the presence of a nucleic acid encoding the        YWHAE-FAM22 fusion protein, if at least one of the fusion        protein or the nucleic acid is detected, then selecting, and        optionally administering, a treatment regimen comprising an        effective amount of an anti-cancer agent.-   20. The assay of paragraph 19, wherein the nucleic acid comprises    the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.-   21. The assay of paragraph 19 or 20, wherein the fusion protein    comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.-   22. A method of treating endometrial stromal sarcoma in a subject in    need thereof, the method comprising:    -   (i) providing a composition comprising a drug to treat        endometrial stromal sarcoma; and    -   (ii) administering an effective amount of the drug to the        subject so as to treat endometrial stromal sarcoma,    -   wherein the subject expresses a nucleic acid encoding a        YWHAE-FAM22 fusion protein.-   23. The method of paragraph 40, wherein the nucleic acid comprises    the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.-   24. An isolated sample obtained from a subject, wherein the sample    comprises a YWHAE-FAM22 fusion protein or a nucleic acid encoding    the fusion protein, and a or a fusion and a first reagent that binds    with the fusion protein or the nucleic acid, and wherein the reagent    is adapted to produce a signal so as to detect presence of the    fusion protein or the nucleic acid in the isolated sample.-   25. The isolated sample of paragraph 24, wherein the sample is    selected from the group consisting of blood, urine, plasma, tissue,    cell, saliva, and any combinations thereof.-   26. The isolated sample of paragraph 24 or 25, wherein the subject    is a mammal.-   27. The isolated sample of any of paragraphs 24-26, wherein the    subject is a human.-   28. The isolated sample of any of paragraphs 24-27, wherein the    first reagent is selected from the group consisting of a nucleic    acid, an antibody, a small molecule, a polypeptide, a peptide, a    lipid, an oligo- or poly-saccharide, and any combinations thereof.-   29. The isolated sample of any of paragraphs 24-28, wherein the    first reagent further comprises a label to produce a signal so as to    detect presence of the fusion gene or the fusion protein in the    isolated sample.-   30. The isolated sample of paragraph 29, wherein the label is    selected from the group consisting of a radiolabel, a chromophore, a    fluorophore, an enzyme, and any combinations thereof.-   31. The isolated sample of any of paragraphs 24-30, wherein the    first reagent is covalently or non-covalently linked to a solid    support.-   32. The isolated sample of paragraph 31, wherein the solid support    is selected from the group consisting of a chip, a microarray, a    gel, a test strip, and any combinations thereof.-   33. The isolated sample of any of paragraphs 24-32, wherein the    sample comprises a second reagent, wherein the second reagent binds    with the first reagent, the fusion protein or the nucleic acid.-   34. The isolated sample of paragraph 33, wherein the second reagent    further comprises a label to produce a signal so as to detect    presence of the first reagent bound to the fusion protein or the    nucleic acid in the isolated sample.-   35. The isolated sample of paragraph 34, wherein the label is    selected from the group consisting of a radiolabel, a chromophore, a    fluorophore, an enzyme, and any combinations thereof.-   36. The isolated sample of any of paragraphs 33-35, wherein the    second reagent is covalently or non-covalently linked to a solid    support.-   37. The isolated sample of paragraph 36, wherein the solid support    is selected from the group consisting of a chip, a microarray, a    gel, a test strip, and any combinations thereof.-   38. The isolated sample of any of paragraphs 24-37, wherein the    nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1 or    SEQ ID NO: 2.-   39. The isolated sample of any of paragraphs 24-38, wherein the    fusion protein comprises the nucleotide sequence of SEQ ID NO: 3 or    SEQ ID NO: 4.-   40. A composition comprising:    -   (i) a YWHAE-FAM22 fusion protein or a nucleic acid encoding the        YWHAE-FAM22 fusion protein, wherein the YWHAE-FAM22 fusion        protein or the nucleic acid is at least partially isolated from        a biological sample obtained from a subject; and    -   (ii) a reagent that binds with the fusion protein or the nucleic        acid, wherein the reagent is adapted to produce a signal so as        to detect presence of the fusion protein or the nucleic acid in        the composition, and wherein the YWHAE-FAM22 fusion protein or        the nucleic acid is in a biological sample obtained from a        subject.-   41. A system comprising:    -   (i) a biological sample obtained from a subject; and    -   (ii) a reagent that binds with a YWHAE-FAM22 fusion protein or a        nucleic acid encoding the YWHAE-FAM 22 fusion protein, wherein        the reagent is adapted to produce a signal so as to detect        presence of the fusion protein or the nucleic acid in the        biological sample obtained from the subject.-   42. A computer system comprising for obtaining data from at least    one test sample obtained from at least one subject, the system    comprising:    -   (i) at least one determination module configured to receive said        at least one test sample and perform at least one analysis on        said at least one test sample to determine the presence or        absence of a YWHAE-FAM22 fusion protein or a nucleic acid        encoding the same;    -   (ii) at least one storage device configured to store data output        from said determination module; and    -   (iii) at least one display module for displaying a content based        in part on the data output from said determination module,        wherein the content comprises a signal indicative of the        presence of the fusion gene or the fusion protein.-   43. An isolated nucleic acid encoding a YWHAE-FAM22 fusion protein.-   44. The isolated nucleic acid of paragraph 43, wherein the isolated    nucleic acid comprises the nucleotide sequence of SEQ ID NO: for SEQ    ID NO: 2.-   45. The isolated nucleic acid of paragraph 43 or 44, wherein the    YWHAE-FAM22 fusion protein comprises the amino acid sequence of SEQ    ID NO: 3 or SEQ ID NO: 4.-   46. An isolated fusion protein, wherein the fusion protein is    encoded by a YWHAE-FAM22 fusion gene.-   47. The isolated fusion protein of paragraph 46, wherein the fusion    protein comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID    NO: 4.-   48. A kit for assessing endometrial stromal sarcoma status in a    subject, the kit comprising at least one reagent adapted for    detecting the presence of a YWHAE-FAM22 fusion gene or a fusion    protein encoded by the fusion gene.-   49. The kit of paragraph 48, wherein the at least one reagent is    anchored on a solid support.-   50. A method of treating endometrial stromal sarcoma in a subject in    need thereof, the method comprising:    -   (i) assaying a biological sample from the patient for presence        of a YWHAE-FAM22 fusion protein or a nucleic acid encoding        thereof; and    -   (ii) if the fusion protein or the nucleic acid is detected in        the sample, administering an anti-cancer therapy to the subject.

The disclosure is further illustrated by the following examples whichshould not be construed as limiting. The examples are illustrative only,and are not intended to limit, in any manner, any of the aspectsdescribed herein. The following examples do not in any way limit theinvention.

EXAMPLES Example 1 14-3-3 Fusion Oncogenes in High-Grade EndometrialStromal Sarcoma

Study Samples:

The study samples include frozen and formalin-fixed paraffin-embeddedtissues retrieved from tumor banks and pathology archives at Brigham andWomen's Hospital, Catholic University of Leuven, Vancouver GeneralHospital, and Stanford University Medical Center with the approval ofthe respective institutional research boards. Cell lines, includingESS1, ESS-JAZFE gastrointestinal stromal tumor (GIST430), andleiomyosarcoma (LMS03), were developed at Brigham and Women's Hospital.

Cytogenetic Analysis and FISH:

Cytogenetic analysis was performed on Giemsa-banded metaphase spreadsper standard protocol (21). FISH analyses were performed on 4-μm tissuesections that were prebaked for 2 h at 60° C. The sections weredeparaffinized in xylene three times for 15 min each and dehydratedtwice in 100% ethanol for 2 min. The slides were immersed in Tris-EDTA[100 mM Tris base and 50 mM EDTA (pH 7.0)] for 45 min at 95-99° C. andrinsed in 1×PBS for 5 min. Proteolytic digestion of the sections wasperformed using Digest-ALL 3 (Invitrogen) at 37° C. for 20 min, twice.The sections were then sequentially dehydrated in alcohol (70%, 85%,95%, and 100%) for 2 min each and air-dried. The YWHAE break-apart probewas composed of two sets of overlapping BAC clones (Children's HospitalOakland Research Institute), telomeric (RP11-143L7 and RP11-22G12,biotin-labeled) and centromeric (RP11-100F18 and RP11-60C18,digoxigenin-labeled), detected with streptavidin Alexa Fluor 594conjugate (Invitrogen) and FITC anti-digoxigenin (Roche Diagnostics).The 10q23.2 (FAM22A region) breakpoint flanking probes were RP11-1005L9(biotin-labeled) and RP11-210E13 (digoxigenin-labeled), and the 10q22.3(FAM22B-region) breakpoint flanking probes were RP11-715A21(biotin-labeled) and RP11-668E21 (digoxigenin-labeled). One hundrednuclei per case were evaluated. Paired signals were defined as an orangeand green signal less than two signal diameters apart or a single yellow(overlapping) signal, whereas unpaired signals were those separated bygreater than or equal to two signal diameters. Only cases with clearlyvisible probe signals observed in at least 100 nuclei were consideredinterpretable. A case was considered to be positive for rearrangement ifunpaired signals were seen in >20% of nuclei.

Paired-End RNA (Transcriptome) Sequencing and deFuse Analysis:

RNA extraction and sequencing were performed as previously described(22-24). Double-stranded cDNA was synthesized from polyadenylated RNA,and the resulting cDNA was sheared. The 190- to 210-bp DNA fraction wasisolated and

PCR-amplified to generate the sequencing library, as per the IlluminaGenome Analyzer paired-end library protocol (Illumina). The resultinglibraries were sequenced on an Illumina GA II. Short read sequencesobtained from the Illumina GA II were mapped to the reference humangenome (NCBI build 36.1, hg18) plus a database of known exon junctions 2by using MAQ 3 in paired-end mode. Gene fusions were predicted withdeFuse (13), which predicts gene fusions by searching paired-endRNA-sequencing data for reads that harbor fusion boundaries. Spanningreads harbor a fusion boundary in the unsequenced region in the middleof the read, whereas split reads harbor a fusion boundary in thesequence of one end. deFuse searched for spanning reads with read endsthat align to different genes. Approximate fusion boundaries implied byspanning reads were then resolved to nucleotide level by using dynamicprogramming-based alignment of candidate split reads.

RT-PCR and Sequencing:

RNAs from frozen tumor and cell line samples were extracted with amirVana miRNA Isolation Kit (Ambion) according to the manufacturer'sprotocol. Reverse transcription was subsequently performed with aniScript cDNA Synthesis Kit to generate cDNA with 1 μg of RNA sample.Forward primers specific for YWHAE (exon 1A: 5′-AGAGGCTGAGAGAGTC GGAGACACTA-3′ (SEQ ID NO: 81); exon 1B: 5′-TATGGATGATCGAGAGGATCTGGTG-3′ (SEQ IDNO: 82); and exon 5: 5′-CAGAAC TGGATACGC TGAGT GAAGAA-3′ (SEQ ID NO:83)) and a reverse primer specific for FAM22A/B (exon 2: 5′-CTCATAGACACTCCTGG GGTTACAGG-3′ (SEQ ID NO: 84)) were used. PCR was performed withPCR SuperMix (11306; Invitrogen) according to the manufacturer'sprotocol with the following cycling conditions: 1 cycle at 94° C. for 2min followed by 30 cycles of 94° C. for 0.5 min, 55° C. for 0.5 min, 68°C. for 2 min, and a final extension of 68° C. for 5 min. PCR productswere evaluated on a 1% agarose gel alongside 1 Kb Plus DNA Ladder(Invitrogen) visualized with ethidium bromide staining. The PCR ampliconbands were excised from the gel, purified with a Qiagen Gel PurificationKit, and sequenced with BigDye Terminator v3.0 Ready Reaction CycleSequencing (Applied Biosystems) on an ABI PRISM 310.

Fusion Construct and Cloning:

YWHAE-FAM22A-FLAG fusion cDNA containing BamHI (YWHAE end) and EcoRI(FLAG end) restriction sites was synthesized (GenScript) based on thesequences of the fusion transcript present in ESS1 and cloned in pUC57vector. The fusion gene sequence was validated by sequencing. It wasfurther subcloned in pCDNA3(+) by EcoRI and BamHI (GenScript). Theconstruct integrity was verified by sequencing. The fusion construct wasexpressed in 293T cells by a Lipofectamine-based transfection methodaccording to the manufacturer's instructions (Invitrogen LifeTechnologies).

Cell Lysate Preparation:

Whole-cell lysates were prepared in lysis buffer [1% Nonidet P-40, 50 mMTris.HCl (pH 8.0), 100 mM sodium fluoride, 30 mM sodium pyrophosphate, 2mM sodium molybdate, 5 mM EDTA, and 2 mM sodium orthovanadate]containing protease inhibitors (10 μg/mL aprotinin, 10 μg/mL leupeptin,and 1 mM phenylmethylsulfonyl fluoride). Nuclear and cytoplasmicfraction lysates were prepared by using a Qproteome Cell Compartment Kit(Qiagen) according to the manufacturer's protocol. Proteinconcentrations were determined by using the Bio-Rad Protein Assay.

Western Blotting and Immunoprecipitation Studies:

Electrophoresis and Western blotting were performed as describedpreviously (25). In short, 30 μg of protein was loaded on a 4-12%Bis-Tris gel (NuPAGE; Invitrogen) and blotted onto a nylon membrane.Immunoprecipitations were performed by incubating 1 mg of preclearedcell lysate with anti-FLAG (mouse monoclonal, F1804; Sigma) for 2 h at4° C., followed by addition of 20 μL of protein A Sepharose (ZymedLaboratories) for overnight incubation at 4° C. The immunoprecipitateswere then washed three times with lysis buffer and one time with 750 μLof 10 mM Tris (pH 7.4) buffer for 10 mM each at 4° C., before beingresuspended in SDS/PAGE loading buffer containing 7.5%β-mercaptoethanol, heated at 95° C. for 5 min, resolved on 4-12%SDS/polyacrylamide gradient gels (NuPAGE; Invitrogen), and transferredto nylon membranes. Adequate protein transfer was demonstrated bystaining the membranes with Ponceau S (Sigma Chemical).

The following primary antibodies were used for staining: antibodiesraised against N-terminal (amino acids 1-70) YWHAE (rabbit polyclonal,HPA008445; Sigma) and against C-terminal (amino acids 239-255) YWHAE(rabbit polyclonal, BML-SA475R; Enzo Life Sciences), anti-FLAG (mousemonoclonal, F1804; Sigma), anti-FOXO3A (rabbit polyclonal, 9467; CellSignaling), anti-poly(ADP-ribose) polymerase (PARP, mouse monoclonal,33-3100; Zymed), and anti-GADPH (mouse monoclonal, G8795; Sigma).Detection was by ECL (Amersham Pharmacia Biotechnology) with a FujiLAS1000 Plus chemiluminescence imaging system.

Preparation of Lentiviral FAM22A shRNA Constructs and LentiviralInfections:

FAM22A shRNAs were from Broad Institute RNAi Consortium: FAM22A shRNA1(NM_001099338.1-3119s21c1), 5′-TCTTGCTGGGCCTTAGCTTTG-3′ (SEQ ID NO: 85);and FAM22A shRNA2 (NM_001099338.1-598s21c1), 5′-TATGTTCCAGGAACCTGTTTA-3′(SEQ ID NO: 86). Lentiviral preparations were produced by cotransfectingempty vector pLKO.1 puro with FAM22A shRNA and helper virus packagingplasmids pCMVΔR8.a91 and vsv-g (at a 10:10:1 ratio) into 293T cells.Transfections were carried out with Lipofectamine and PLUS reagent.Lentiviruses were harvested at 24, 36, 48, and 60 h posttransfection.Viruses were frozen at −80° C. in aliquots at appropriate amounts forinfection. ESS1 cells were seeded in 6-well plates. Infections werecarried out in the presence of 8 μg/mL polybrene. After transduction,ESS1 were selected with 2 μg/mL puromycin for 15 d, then lysed forWestern blot analysis. Cell culture images were obtained by using a SpotRT Slider Camera and Spot software (Version 4.6 for Windows) and a NikonEclipse TE2000-S inverted microscope.

In Vitro Wound-Healing Assays:

Cell-wounding studies were carried out via standard methods (26). Aslash was created in confluent cell cultures, using the tip of a P-100Pipetman, at 8 d after shRNA transduction with puromycin selection. Theplates were photographed at 0, 72, and 96 h with Spot software (Version4.6 for Windows) and a Nikon Eclipse TE2000-S inverted microscope.

3′ End Sequencing Gene-Expression Analysis:

We prepared 3′ sequence libraries as previously described (27). TotalRNA was purified from formalin-fixed paraffin-embedded sections afterdeparaffination with a xylene incubation, ethanol wash, andprotease/DNase digestion (RecoverAll Total Nucleic Acid Isolation Kit;Ambion) per the manufacturer's protocol. Isolation of the mRNA 3′ endswas achieved by oligo(dT) selection on 20 μg of total RNA with theOligotex mRNA Mini Kit (Qiagen). Insufficiently fragmented RNA washeat-sheared to ˜100-200 bp. The poly(A)-selected RNA was then subjectedto first- and second-strand cDNA synthesis and Illumina librarysynthesis. To obtain 36-base single-end sequence reads, 3′-endsequencing for expression quantification (3SEQ) libraries were sequencedwith Illumina GA IIx machines. Reads were mapped first to thetranscriptome (refMrna, downloaded from the UCSC genome browser atwww.genome.ucsc.edu) by using SOAP2, allowing at most two mismatches(28). Unmapped and nonuniquely mapping reads were then mapped againstthe human genome (hg19), also using SOAP2, and reads mapping to RefSeqexons (same strand) were determined. Total sequence reads for each genesymbol from the transcriptome mapping and genome mapping were summed tocreate the gene-expression profile matrix. The data were then normalizedby expressing the number of reads as transcripts per million reads (TPM)and filtered to select genes with a value of ≧1 TPM in at least twosamples and an absolute difference of ≧2 TPM across the series. Fromthese genes, those with an SD≧200 as determined by Cluster 3 softwarewere log-transformed, centered by gene using Cluster 3 software,subjected to unsupervised hierarchical clustering by Centroid linkage,and visualized with Java TreeView. Significance analysis of microarrays(SAM; www-stat.stanford.edu/˜tibs/SAM/) was used to identify genesexpressed differentially between the tumor groups.

siRNA Study and Cell Viability Assay.

According to the manufacturer's instructions, transfections were carriedout with Lipofectamine and PLUS reagent (Invitrogen Life Technologies).Briefly, scrambled control (5′-AAGUUCAGGUCGAUAUGUGCA-3′ (SEQ ID NO: 87);Invitrogen Life Technologies) or FAM22 siRNAs (s198355 and s195919;Invitrogen Life Technologies) incubated with PLUS in serum-free mediumfor 15 min at room temperature, then mixed in diluted Lipofectamine inequal volume with scrambled control or siR-NAs-PLUS mixtures andincubated for another 15 min at room temperature. Finally,siRNA-PLUS-Lipofectamine complexes were added into 60% confluent ESS1cells under serum-free medium conditions in 6- or 96-well plates.DNA-PLUS-Lipofectamine complexes in serum-free medium were completelyreplaced with serum-containing regular medium after a 3-h incubation.Cells were lysed for Western blot analysis at 96 h posttransfection, andcell viability was determined after 96 h posttransfection with theCellTiter-Glo luminescent assay from Promega. The viability data werenormalized to the scrambled control group. All assays were performed inquadruplicate wells and averaged from two independent transfections inESS1 cells.

Quantitative Cell Migration Assay.

Transfections of NIH 3T3 cells were carried out with Lipofectamine andPLUS reagent(Invitrogen Life Technologies) according to themanufacturer's protocol. At 24 h posttransfection, 0.5 mL of serum-freemedia containing 5×10⁴ NIH 3T3 cells was plated per BD BioCoat8.0-μm PETMembrane 24-well Cell Culture Insert (no. 354578; BD Biosciences). Next,the wells were fed with 0.75 mL of Iscove's modified Dulbecco's mediumcontaining 15% FBS and incubated in a humidified incubator at 37° C., 5%CO₂ for 60 h. The media from the inside of the insert was aspirated, andthe interiors of the inserts were gently swabbed to remove nonmigratorycells. Inserts were transferred to new wells containing 400 μL, of CellStain Solution (no. 11002; Cell Biolabs) and incubated for 10 min atroom temperature, then rinsed two times in a beaker of water. Then theinserts were air-dried, imaged with a scanner, and quantified with amicro-plate reader.

Results and Discussion

Cytogenetics and Whoe-Transcriptome Sequencing Identifies YWHAE-FAM22A/BFusion as a Frequent Recurrent Genetic Event in High-Grade ESS

To characterize the genetic basis of high-grade ESS, we performedprospective cytogenetic G-banding analyses, which identified atranslocation, t(10;17)(q22;p13), as a recurrent and predominantaberration in 7 of 12 cases (FIG. 1A and Table 1). A spontaneouslyimmortal cell line, ESS1, was established from one of theset(10;17)-bearing ESS. Fluorescence in situ hybridization (FISH)localized the ESS 17p13 translocation breakpoint to the YWHAE (14-3-3c)gene (FIG. 1B). In contrast to the tumor cells, the adjacent normalmyometrial tissues uniformly lacked YWHAE rearrangement by FISH,confirming the somatic nature of the rearrangement. One ESS had anunbalanced t(10;17), associated with deletion of the rearranged YWHAE 3′end, thereby implicating the YWHAE 5′ end in a putativet(10;17)-associated fusion oncogene. FISH localizations mapped the 10qtranslocation breakpoint, in each t(10;17) ESS, to one of two regions(10q22.3 and 10q23.2) separated by 7.8 megabases (FIG. 5): notably,these regions had gnomic and organizational similarities, eachcontaining two members of the FAM22 family. FISH mapping within theseregions was hampered by the repetitive nature of the genomic sequences(FIG. 5). Because of the abundant expression of wild-type YWHAE, 3′ RACEanalysis was unsuccessful.

TABLE 1 Karyotypes of 12 histologically high grade endometrial stromalsarcomas Case number Karyotype 1 46, XX, t(10; 17)(q22; p13) 2 46, XX,t(10; 17)(q22; p13) 3 43, XX, der(5)t(5; 21)(q35; q11), der(9; 11)(q10;q10), −10, t(10; 17)(q22; p13), −21 4 55-58, XX, del(X)(p11.2), +1,i(1)(q10), +2, +3, +4, +6, del(6)(q21), +7, −9, +12, del(12)(q21), +15,+17, +22, add(22)(q12) × 2, +2r 5 44, XX, t(10; 17)(q22; p13),del(11)(q1?2), −19, −22 6 46, XX, inv(6)(p21q13)[10] 7 46, XX,del(X)(p22.1), +1, ?dup(1)(q42), i(1)(q10), +2, +3, +4, t(4; 7)(q21;p22), +7, −9, +12, +17, der(17)t(5; 17)(p11; p11), +22, add(22)(q13) ×2, +2-4mar 8 46, X, der(X)t(X; 1)(p22; q24), dup(1)(q12q32) 9 45, X, −X,t(10; 17; 12)(q22; p11.2; q13), add(19)(p13.3) 10 47, XX,der(9)del(9)(p11)del(9)(q12), del(10)(q22), der(11)t(9; 11)(q12; q12),der(17)t(10; 17)(q22; p13), +19 11 47, XX, +i(1)(q10), t(9; 9)(p24;q11), add17(p13), −16, +mar 12 46, XX, t(10; 17)(q22; p13)

To demonstrate a putative YWHAE fusion oncogene in these genomicallyrepetitive 10q regions, we used whole-transcriptome sequencing as anunbiased method. Sequencing was performed against thet(10;17)-containing, ESS1, and sequence reads were analyzed by using acustom-written defuse algorithm designed to identify fusion transcriptsin RNA sequencing datasets (13), including those involving members ofhighly homologous gene families. deFuse analysis identified in-frameYWHAE-FAM22A fusions of YWHAE exon 5 to FAM22A exon 2 (FIG. 1C and Table2). FAM22A is located within the 10q23.2 breakpoint region, whereas thealternate breakpoint region, 10q22.3, contains F4M22B (encoding aprotein with 99% amino acid identity to FAM22A) and FAM22L RT-PCR withYWHAE forward primers and consensus reverse primers for FAM22A/B/Eidentified YWHAE-FAM22B fusion transcripts in each t(10;17) ESS thatlacked YWHAE-FAM22A (FIG. 1D). Therefore, FAM22A and FAM22B arealternative YWHAE gene fusion partners (FIG. 1E). In all cases, thegenetic rearrangements in transcribed YWHAE-FAM22 involved fusion ofYWHAE exon 5 to FAM22A or FAM22B exon 2, creating a fusion codingsequence consistent with genomic breakpoints in YWHAE intron 5 andFAM22A/B intron 1. FAM22A and FAM22B have sequence homology with NUT, anoncogene fused to BRD4 and BRD3 bromodomain genes in NUT midlinecarcinoma (14, 15). The YWHAE-FAM22A fusion transcript is 2,970 bp inlength, and the corresponding protein product contains 989 aa, with apredicted molecular mass of 108 kDa (SEQ ID Nos: 1-4 and GenBankaccession nos. JN999698 and JN999699)

TABLE 2 Summary of the result of deFuse analysis in ESS1 (including onlyfusion transcripts with >0.9 prediction probability and sorted bytranscript count). Split transcript breakpoint Coding Coding ExonicExonic Expression Expression Gene_name 1 Gene_name 2 count homology 1 2deletion eversion 1 2 1 2 FAM22A YWHAE 76 1 Y Y N N Y Y 340 9303 BIRC1AC139834.2 63 1 Y N N N Y N 8850 88 KIAA1267 ARL17 49 4 Y Y N Y Y Y 38711792 ARL17P1 KIAA1267 47 4 Y Y N Y Y Y 1863 3871 IGLV5-52 BMS1 34 4 Y YN N Y Y 29 1976 KIAA1267 ARL17P1 34 4 Y Y N Y Y Y 3871 1863AL159167.23-1 C9orf102 28 66 Y Y Y N Y Y 190 644 GTF2H2 BIRC1 26 2 Y Y NY Y Y 1374 8850 BIRC1 AC140134.2-2 25 2 Y Y N Y Y Y 8850 3264 BACH1AF124731.2 24 2 Y Y Y N Y Y 3428 19 C18orf32 RPL37P33 20 2 Y Y N N Y Y1765 9711 RMND5A ANAPC1 19 3 Y Y N N Y Y 1371 1945 IFNGR2 TMEM50B 18 2 NN Y N N N 1463 3079 D87018.1-3 IGLV5-52 16 4 N Y Y N N Y 8 29 CKMT2ZCCHC9 15 2 N N Y N N N 139 712 AC103702.3 HOXB5 14 1 Y N Y N Y N 28 124AC145138.2-1 GUSBL2 14 5 Y Y N N Y Y 49 158 MRPS5 ZNF514 13 1 N N Y N NY 979 1235 TSHZ2 SLC35A1 11 2 N N N N N N 2950 959 AC010326.7-2 ZNF58711 3 Y N Y N Y N 135 3298 FBXO25 BET1L 10 4 Y Y N N Y Y 506 773 SLC25A6IL3RA 10 2 Y Y Y N Y Y 6782 122 MTHFD1 ZBTB25 10 2 Y Y Y N Y Y 856 302DPYSL2 PNMA2 10 3 Y N Y N Y N 4132 3142 MTO1 EEF1A1 10 2 N Y Y N N Y 662527400 KPNA2 SNRNP200 10 0 Y Y N N Y Y 680 10065 ANKDD1A SPG21 10 8 Y YY N Y Y 281 2825 GTF2H2 BIRC1 8 2 N Y N Y N Y 1374 8850 AC024270.6TM6SF1 7 2 Y N Y N Y N 1817 105 EIF3CL PDXDC2 7 4 Y N N Y Y Y 3865 2617YY1 SLC25A29 7 2 N N Y N N Y 1840 1521 GOLGA7B CRTAC1 7 2 N Y Y N N Y387 1889 CSAD ZNF740 7 3 N Y Y N N Y 1881 1069 GP1BA CHRNE 6 10 Y Y Y NY Y 178 452 FALZ ARL17 6 2 Y Y N N Y Y 8711 1792 BTBD7 OPTN 6 3 Y N N NY N 2154 255 YARS2 DNM1L 6 0 Y N Y N Y N 510 3557 B9D1 EPN2 6 10 Y N Y NY N 106 3274 DNAJC2 PMPCB 6 188 N Y Y N N Y 862 2433 FBXW2 AL161911.17 54 N Y Y N N Y 3984 114 EGLN2 CYP2F1 5 2 Y Y N N Y Y 588 9 SAV1 GYPB 5 7Y Y N N Y Y 984 163 DNAJB4 FUBP1 5 2 Y N Y N Y N 1702 4211 VPS45 PLEKHO15 3 Y Y Y N Y Y 1676 334 LRRC37A NSF 5 0 Y Y N Y Y Y 1435 2154 HEXABRUNOL6 5 4 Y N Y N Y N 2233 544 PRKAA1 TTC33 4 2 Y Y Y N Y Y 4151 1190ADSL SGSM3 4 3 Y Y Y N Y Y 1390 905 RPS23 ATG10 4 3 Y Y Y N Y Y 28000198 EGLN2 CYP2F1 4 2 Y Y N N Y Y 588 9 USO1 G3BP2 4 3 N Y Y N N Y 46125924 USP45 SFRS18 4 2 N N Y N N N 1083 7585 PEX13 KIAA1841 4 10 Y Y Y NY Y 654 384 AL133216.10-2 PCMTD2 4 2 N Y N N N Y 598 4820 AC138894.2-2PDXDC2 4 376 Y N Y N Y Y 26 2617 AC073135.3-3 LMLN 3 3 N N Y N Y N 93220 ENTPD7 COX15 3 2 N Y Y N N Y 502 2450 CENPB CDC25B 3 3 N Y Y N N Y1088 1274 GKAP1 KIF27 3 3 N Y N Y Y Y 373 941 SLC12A7 NKD2 3 2 N Y Y N NY 1529 887 ENPP3 CRSP3 3 0 Y N Y N Y N 2424 2947 DHTKD1 SEC61A2 3 2 Y YY N Y Y 1228 776 TMEM14B MAK 3 3 Y Y Y N Y Y 1378 126 RPL24P6AC084198.31-2 3 6 Y N Y N Y Y 13085 67 IL17D N6AMT2 2 3 N N Y N N N 191320 ZNF649 ZNF577 1 2 Y N Y N Y Y 617 669 FOXO3B Z95118.1 1 3 Y Y N N YY 103 7 CLSTN1 CTNNBIP1 1 1 Y Y Y N Y Y 12529 976 AC005488.2-6AC005488.2-4 1 3 N N Y N Y Y 258 202 REXO4 ADAMTS13 1 0 N Y Y N N Y 428277 Expression 1 and Expression 2 represent the total number of readsaligned uniquely to Gene 1 and Gene 2 respectively, while the Splittranscript count represents the number of split reads where the fusionsequence aligns to each of these genes or to an indistinguishable familymember (a measure that facilitates the identification of fusionsinvolving genes with highly homologous family members). OnlyYWHAE-FAM22A fusion (highlighted in yellow) was experimentally testedand validated.

YWHAE-FAM22 is Expressed in t(10;17)-Bearing High-Grade ESS andDemonstrates Transforming Properties.

To identify expression of YWHAE-FAM22A and YWHAE-FAM22B, Westernblotting was performed with N-terminal and C-terminal YWHAE antibodies,of which only the N-terminal antibody was expected to recognize thefusion proteins. Although both antibodies identified ˜30-kDa wild-typeYWHAE in all tumor samples examined, only the N-terminal YWHAE antibodyidentified putative YWHAE-FAM22A/B fusion proteins, which wererepresented in each t(10;17) ESS by bands at 110 kDa and 140 kDa (FIG.6). The 110-kDa form corresponds to the predicted molecular mass forYWHAE-FAM22A/B, whereas the 140-kDa form presumably represents a matureform of the fusion protein, after posttranslational modifications.YWHAE-FAM22A/B expression was considerably lower than that of the nativeYWHAE, in keeping with the whole-transcriptome sequence data that showedeight times fewer YWAE-FAM22A reads than wild-type YWHAE reads in thebreakpoint region. YWHAE-FAM22A/B oncoproteins were not detected in ESSor other sarcomas lacking t(10:17) nor were they detected in t(10:17)ESS by using antibodies to the YWHAE C-terminal region. Furthermore,endogenous ESS YWHAE-FAM22A/B fusion proteins comigrated with aFLAG-tagged YWHAE-FAM22A pcDNA3 construct expressed in HEK 293T cells(FIG. 6). These studies demonstrated equivalent YWHAE-FAM22A/Bexpression levels in t(10;17) ESS biopsy specimens compared with theESS1 immortal cell line.

YWHAE-FAM22A oncogenic roles were evaluated in t(10;17) ESS1 cells byusing shRNAs and siRNAs targeting FAM22A. FAM22A shRNA1 targets exon 2,which is contained in the fusion transcript. A control sequence, FAM22AshRNA2, targets exon 1, which is not in the fusion transcript, and isexpected to inhibit wild-type FAM22A/B/D/E. The nonfusion transcript isminimal to absent in virtually all adult tissues and cancers(www.ncbi.nlm.nih.gov/sites/entrez?db=unigene), and ESS1whole-transcriptome sequencing showed that only 3% of reads in thebreakpoint region were wild-type (unrearranged) FAM22A, whereas 97% werefusion YWHAE-FAM22A, indicating that wild-type FAM22A is expressed atlow levels in ESS1. In contrast to empty vector and shRNA2, geneknockdown with shRNA1 inhibited YWHAE-FAM22A expression (110- and140-kDa forms) in ESS1, with a corresponding reduction in viability andmigration (FIG. 7). Similarly, ESS1 transfection with siRNAs targetingFAM22A exons 2 or 7 inhibited YWHAE-FAM22A expression, withcorresponding reduction in ESS1 cell viability (FIG. 8). YWHAE-FAM22Atransforming activity was further evaluated in mouse embryonicfibroblast 3T3 cells, where YWHAE-FAM22A but not YWHAE transfectioninduced cell viability and migration (FIGS. 2A-2C).

YWHAE-FAM22 Maintains 14-3-3 Binding Properties and Shows AberrantNuclear Localization.

Structurally, the YwHAE-FAM22A/B oncoproteins contain an intact YWHAEprotein-interaction domain (16), and loss of the YWHAE C-terminal end(encoded by YWHAE exon 6) and fusion to FAM22A/B are not predicted tofunctionally impair this rigid YWHAE protein-interaction domain or itsability to dimerize (FIG. 2D). Further analysis of FAM22A/B proteinsequences revealed a bipartite nuclear localization sequence (Arg-805 toArg-822) encoded by exons 7 of FAM22A and FAM22B. In contrast to nativeYWHAE protein, which is predominantly cytoplasmic (17), YWHAE-FAM22A/Bwas predicted to be predominantly nuclear (18-20). YWHAE-FAM22A/Bnuclear localization was confirmed in ESS1 (FIG. 3A) and in 293T cellsexpressing a YWHAE-FAM22A construct (FIG. 3B).

YWHAE-FAM22 ESS Display Higher-Grade Histology and More AggressiveClinical Course Compared with JAZF1-Rearranged ESS.

Histologically, the 12 clinical cases YWHAE-FAM22A/B ESS (Table 3)exhibited high-grade cytologic features compared with classicnon-t(10;17) ESS (FIG. 4A). In contrast to JAZF1-rearranged ESS, whichdisplayed uniform small round/oval nuclei and low proliferation rate (<5mitotic figures per 10 high-power fields), YWHAE-FAM22A/B ESS showedenlarged nuclei with more irregular nuclear contour and highproliferation rate (>10 mitotic figures per 10 high-power fields).Gene-expression profiling by 3′ mRNA sequencing demonstrated adistinctive expression profile in YWHAE-FAM22A/B ESS compared withJAZF1-rearranged ESS and uterine leiomyosarcoma (FIG. 4B). Genesinvolved in the regulation of cell proliferation (CCND1 and CEBPA) andtissue invasion (MMP15, FSCN1, and TIMP1) were up-regulated inYWHAE-FAM22A/B ESS compared with JAZF1-rearranged ESS (Table 4).Clinically, patients with YWHAE-FAM22A/B ESS presented with higher-stagedisease and experienced more frequent disease recurrence compared withpatients with JAZF1-rearranged ESS (FIGS. 4C and 4D). FISH analysisdemonstrated absolute diagnostic specificity of YWHAE-FAM22A/Brearrangement for high-grade ESS (Table 5). In addition, YWHAE-FAM22A/Brearrangement and JAZF1 rearrangement were mutually exclusive, andYWHAE-FAM22A/B rearrangement was not found in low-grade ESS (n=38) or invarious uterine and nonuterine mesenchymal tumors (55 tumor types,n=827) (Table 6). These findings show that. YWHAE-FAM22A/B rearrangementdefines ea group of uterine sarcomas that is genetically,histologically, and clinically distinct from classic JAZF1-rearrangedESS. This evidence prompts reconsideration of the current classificationof endometrial sarcomas. In the present study, we refer to thisgenetically unique subgroup as YWHAE-FAM22:41.8 ESS. An alternativeclassification consideration would be “14-3-3 ESS,” which has theadvantage of brevity while reflecting the expected biologicalcontributions of YWHAE dysregulation. A biologic classification seemspreferable to “high-grade ESS,” which misleadingly suggest biologiccontinuum with the genetically distinct JAZF1 low-grade ESS.

TABLE 3 Summary of clinicopathologic features of 12 YWHAE-FAM22A/B ESS.FISH/RT- Case Age Tumor examined Histology Clinical stage Follow-up PCR1 67 Primary uterine High grade FIGO stage Alive with disease YWHAE-tumor ESS 1C FAM22A 2 45 Primary uterine High grade FIGO stage Noevidence of YWHAE- tumor ESS 4B disease FAM22A 3 43 Metastatic Highgrade not available not available YWHAE- tumor (lung) ESS FAM22B 4 47Metastatic High grade FIGO stage Alive with disease YWHAE- tumor (lung)ESS 1A FAM22B 5 62 Primary uterine High grade FIGO stage 3 Alive withdisease YWHAE- tumor ESS FAM22B 6 54 Primary uterine High grade FIGOstage Alive with disease YWHAE- tumor ESS 4B FAM22B 7 49 Primary uterineHigh grade FIGO stage Died of disease YWHAE- tumor ESS 4B FAM22B 8 49Primary uterine High grade FIGO stage not available YWHAE- tumor ESS 3BFAM22B 9 57 Primary uterine High grade FIGO stage Died of disease YWHAE-tumor ESS 3C FAM22B 10 28 Primary uterine High grade FIGO stage 3 Alivewith disease YWHAE- tumor ESS FAM22B 11 66 Metastatic High grade FIGOstage Alive with disease YWHAE- tumor (vagina) ESS 3B FAM22B 12 50Primary uterine High grade FIGO stage Alive with disease YWHAE- tumorESS 3C FAM22B

TABLE 4 Filtered 3′ sequencing gene expression dataset of 3YWHAE-FAM22A/B ESS (2 YWHAE-FAM22A and 1 YWHAE-FAM22B), 4JAZF1-rearranged ESS and 4 uterine leiomyosarcomas (LMS); Genes marked $and # are shown to be significantly upregulated and downregulated inYWHAE-FAM22A/B ESS compared to JAZF1-rearranged ESS respectively by SAManalysis (*Numbering for YWHAE-FAM22 ESS corresponds to that in Table3). Tumor YWHAE- YWHAE- YWHAE- JAZF1- JAZF1- JAZF1- JAZF1- FAM22A-FAM22A- FAM22B- ESS-1 ESS-2 ESS-3 ESS-4 ESS-1* ESS-2* ESS-9* LMS-1 LMS-2LMS-3 LMS-4 Sample code STT55 STT55 STT55 STT5543- STT577 STT577 STT577STT58 STT5836_ STT58 STT5853_ 19_ESS 21_ESS 20_ESS ESS 4b_ESS 5b_ESS6b_ESS 37_LMS LMS 38_LMS LMS SORBS3 0.059797 0.009878 0.018274 −0.065952−0.016628 −0.026442 −0.074921 −0.004124 0.021913 0.082789 −0.003926FAM176B 0.033249 −0.023256 0.01193 −0.020513 0.050515 −0.055492−0.043038 0.035408 −0.022771 0.089738 −0.052861 NENF 0.054074 −0.0441140.025917 0.005846 0.043546 −0.075238 −0.076567 0.012926 −0.0118970.032275 0.044347 EFEMP2 0.074317 0.013268 0.051475 0.007135 0.042017−0.044807 −0.086809 −0.00053 −0.020536 0.032228 −0.03381 H19 −0.0558390.02541 0.017175 −0.045044 0.004234 −0.02173 −0.020583 −0.0481060.028759 0.110246 0.003939 PLXNB2 −0.077779 0.046134 −0.011402 −0.0703340.015327 −0.026809 0.015132 −0.025349 0.018752 0.079888 0.022329 SLC16A3−0.003366 −0.006766 −0.027092 0.004614 0.081753 −0.031272 −0.0388110.074324 −0.028365 0.034675 −0.069291 IGFBP6 −0.033425 0.000504 0.0034710.027698 0.061208 −0.052393 −0.064994 0.087897 0.012947 −0.003846−0.042327 FEV −0.007776 −0.040585 0.02796 0.054097 0.099795 −0.061802−0.055832 0.000858 SERPING1 −0.096042 0.038448 0.017024 0.0135840.094589 −0.037827 −0.024647 −0.004868 −0.00528 0.008881 0.004456 LRP1−0.115917 0.013242 0.03215 0.004714 0.063736 0.003168 −0.046153 0.03323−0.002071 −0.010507 0.014938 PLD3 −0.100023 −0.00347 −0.019902 −0.0230410.05819 −0.004599 −0.058881 0.019997 0.055877 0.020103 0.023838 TMSB4X−0.072788 −0.022875 0.010781 −0.054802 0.025333 0.054146 −0.0807110.030212 0.004144 0.033359 0.035229 TNFRSF1A −0.024952 0.015254 0.042736−0.000647 0.048048 −0.020955 −0.11603 0.031554 −0.022929 0.0413060.013851 OLFML3 −0.044779 −0.005322 0.044298 0.00991 0.064265 0.019466−0.102038 0.004916 −0.039003 0.010053 0.040632 CALR −0.096163 0.003893−0.032501 0.053096 −0.009932 −0.013189 −0.056409 0.046221 0.0050660.030924 0.051006 FADS2 −0.055537 0.019421 −0.059798 0.040268 0.055233−0.076052 −0.017312 0.004364 0.004382 0.0644 0.018269 CENPB −0.021679−0.063408 −0.007057 0.0329 0.023943 −0.013565 −0.039215 −0.0226640.116358 −0.001033 −0.018455 SLC44A2 −0.049916 −0.076067 0.0206960.010913 0.034516 0.040931 −0.027354 −0.059368 0.035926 0.069635−0.014427 LAMB2 −0.036039 −0.047236 0.044881 −0.009938 0.03664 −0.018399−0.030012 −0.083109 0.043625 0.045675 0.056491 NFIX −0.066665 −0.085265−0.006692 0.021837 0.014674 −0.031663 −0.006948 0.013051 0.055121−0.009847 0.074194 CD97 −0.032893 −0.041872 −0.014233 0.026608 0.011955−0.050102 −0.028108 −0.040984 0.053065 0.006296 0.102899 GPX4 −0.021626−0.038518 −0.079016 −0.019349 0.01403 0.000948 0.084671 −0.0237430.018025 0.066708 −0.032213 SMARCA4 −0.016639 0.009872 −0.053035−0.040387 −0.030029 −0.00337 0.090064 −0.046043 0.050626 0.053348−0.030671 FAM40B −0.057048 −0.028759 −0.01105 −0.004621 −0.034589−0.051513 0.084565 0.017196 0.008798 −0.018295 0.079865 LOC388692−0.01246 −0.049236 −0.062156 −0.015845 −0.081561 0.001612 0.06690.021337 0.045066 0.0445 0.004647 INADL 0.016974 −0.009705 −0.038868−0.016361 −0.082972 −0.062361 0.082482 0.025338 0.031879 0.0233480.019282 SENP5 −0.025934 −0.018799 −0.031251 0.000365 −0.06728 −0.0499620.106789 0.021244 0.03412 0.00114 0.016316 RNF168 −0.031886 −0.02442−0.032561 0.001333 −0.069201 −0.053214 0.095877 0.035832 0.0262680.006253 0.028484 KCNQ1OT1 −0.035569 −0.024904 −0.035461 −0.020955−0.070279 −0.039187 0.095563 0.040393 0.019899 0.016376 0.028768 NKX3-1−0.047877 0.006683 −0.010597 −0.002002 −0.068665 −0.062286 0.0947410.033836 0.013288 0.00656 0.030405 LOC90784 0.014049 −0.021807 −0.035046−0.003824 −0.073053 −0.034957 0.109765 −0.010157 0.04081 0.0034080.003031 FANCD2 −0.025952 −0.013649 −0.037144 −0.008902 −0.067062−0.023794 0.113405 0.005192 0.045105 −0.001077 −0.002626 DSEL −0.012821−0.024597 −0.043936 −0.013153 −0.06349 −0.033362 0.111573 0.017070.03734 0.011422 −0.005563 PAN3 −0.025747 −0.010211 −0.035949 0.00627−0.0555 −0.043061 0.117554 0.004344 0.030323 −0.014673 0.017504 TMED10P1−0.022041 −0.018328 −0.043225 0.008685 −0.053579 −0.034583 0.122490.000032 0.020878 0.003166 0.005507 COL18A1 0.011312 −0.032113 0.0138630.016029 0.003139 0.019581 0.054575 −0.026556 −0.020393 0.073649−0.103861 AGRN −0.007534 −0.064196 −0.014585 0.027731 −0.007612 0.022008−0.007806 0.037365 −0.019268 0.094107 −0.076981 PLXND1 −0.040151−0.030116 −0.024362 0.02416 −0.011163 0.02834 0.034757 0.003563 0.0124440.084488 −0.095784 THY1 0.035182 −0.028835 −0.002034 −0.006274 −0.063991−0.025295 −0.012615 0.042123 −0.042962 0.10783 −0.007643 FOXS1 0.024104−0.064503 −0.005325 −0.045984 −0.040829 −0.008038 0.042621 −0.0091210.010106 0.10478 −0.026748 PGF 0.033894 −0.069357 −0.010648 −0.010914−0.036165 −0.003138 0.025825 −0.054098 0.002221 0.105485 0.008796 COX4I20.005325 −0.061333 −0.023198 0.001645 −0.071287 0.008745 0.025847−0.004497 −0.014492 0.109047 0.005794 ATP5D 0.021524 −0.05817 −0.0459720.032594 −0.035664 −0.068121 0.020994 −0.005292 0.034984 0.0873080.007856 CRIP1 0.012317 −0.035067 0.024202 0.025094 −0.104521 0.0114690.057318 0.013919 −0.029391 0.058897 −0.029112 PPP1R14A −0.0114370.040546 −0.008174 0.018046 −0.077908 −0.033878 −0.018167 −0.0559880.058047 0.028301 0.07275 APOE 0.00796 0.017037 0.116499 −0.01217−0.048142 −0.053163 −0.026127 0.025269 0.035672 −0.018019 −0.013277TNFRSF12A −0.013217 0.040729 0.023507 −0.056819 −0.045596 0.010861−0.030686 0.088194 0.036951 −0.001766 −0.066428 MXRA8 −0.017502 0.0075460.031067 0.00974 0.009263 −0.037413 −0.043456 0.106046 0.028118−0.069125 −0.026468 CREB3L1 −0.020386 −0.007099 0.038221 −0.025746−0.018011 −0.072863 −0.027 0.098324 0.055565 −0.023863 −0.005975 COL1A2−0.00309 −0.052719 0.037229 −0.027419 −0.016961 −0.029998 −0.0480510.09506 0.058155 0.004756 −0.038056 COL1A1 −0.00055 −0.049258 0.065757−0.011898 −0.00873 −0.035728 −0.052192 0.08821 0.047655 −0.022967−0.02764 SRPX2 0.050878 −0.003649 0.025543 −0.022956 −0.044629 −0.049248−0.04987 0.103912 0.018444 −0.015108 −0.016148 TCIRG1 0.040481 0.038197−0.006229 −0.016383 −0.029963 −0.077162 −0.034229 0.069094 0.0483490.028226 −0.054551 CAPG 0.002851 0.011206 0.01251 0.016549 −0.061517−0.058081 −0.015747 0.095269 0.044258 0.010579 −0.05653 TGFB1 0.003953−0.004578 −0.019087 −0.02361 −0.06624 −0.032472 −0.015549 0.0805840.069517 0.039028 −0.053109 CTSD 0.040317 −0.00223 −0.029601 0.016242−0.03172 −0.051523 −0.066664 0.083205 0.059009 0.005628 −0.031627 SEPN1−0.013153 0.024349 0.012897 0.022947 0.007019 −0.048225 −0.0697220.067045 0.055962 0.020899 −0.074525 COL7A1^(#) 0.035743 0.0031 0.0402890.050596 −0.019648 −0.039844 −0.03899 0.073303 0.032677 −0.050309−0.066152 ACAP3 0.00094 0.061976 −0.002387 0.015675 −0.08018 −0.0316170.011559 0.035799 0.076142 −0.055458 −0.022407 DDAH2 0.047713 0.067199−0.070161 0.013474 −0.02211 0.003872 −0.087351 0.043346 −0.0157850.010285 0.006939 P4HB −0.007837 0.040766 −0.053875 −0.002424 0.024914−0.015301 −0.063536 0.108457 −0.008465 −0.014924 −0.027048 FKBP2−0.002926 0.045107 −0.019178 −0.049744 0.032621 −0.028751 −0.1013150.066147 0.001787 0.025115 0.016194 ARF5 0.01108 0.00956 −0.062292−0.018196 −0.04209 0.004583 −0.090861 0.061287 0.053923 0.0334550.007442 HNRNPUL2 0.000639 −0.021647 −0.029308 −0.012468 −0.04367−0.009243 −0.092301 0.059278 0.0612 0.055196 0.003305 PLOD3 −0.02130.030594 −0.065921 0.007522 −0.037084 −0.038128 −0.068305 0.0771330.030739 0.041336 0.020561 EMILIN1 −0.010523 0.00439 −0.02214 0.010677−0.021287 −0.062741 −0.074439 0.085117 0.05879 −0.01134 0.028595 RABAC1−0.019451 0.015281 −0.029869 0.008479 −0.025506 −0.033519 −0.0686690.045526 0.108958 −0.017385 −0.000415 SRC −0.028529 0.013502 −0.034919−0.000225 −0.017886 −0.069782 −0.062435 0.017129 0.066342 0.0724250.032697 ZYX −0.002767 0.037931 0.00933 −0.055467 −0.053707 −0.035978−0.067974 0.024461 0.0627 0.005539 0.065899 HSPB1 −0.028153 0.027785−0.072178 −0.046169 0.004242 −0.009025 −0.0652 0.013022 0.07531 0.0087220.056024 ILK −0.041556 0.005801 −0.027143 −0.071046 −0.002648 −0.022895−0.060622 0.028182 0.073998 0.018599 0.062813 GOLGA2 −0.015968 0.053878−0.048071 −0.038201 −0.051613 0.012926 −0.046776 0.055397 0.062230.036718 −0.045561 INPPL1 −0.021003 0.075209 −0.06697 −0.01208 −0.042439−0.038505 −0.046014 0.022983 0.060299 0.042648 0.014143 ANKRD13D0.015213 0.090661 −0.085861 −0.011514 −0.036925 −0.02763 −0.0396410.017613 0.037027 0.031076 0.003428 STIP1 0.00652 0.095834 −0.083511−0.021685 −0.041528 −0.000302 −0.039204 0.019242 0.029504 0.032668−0.00848 IFI27 −0.021783 0.076725 −0.021318 −0.05663 −0.04454 0.002394−0.023084 −0.000702 0.09711 −0.001531 −0.018276 OAZ2 −0.03411 0.001839−0.072312 0.001661 −0.035996 0.04077 −0.052181 0.006978 0.0340480.093546 −0.016558 CLIC1 −0.047599 0.015059 −0.063013 −0.000352−0.035925 0.027289 −0.063721 0.063846 0.04252 0.055072 −0.02892 OAZ1−0.001077 0.017966 −0.110875 −0.043504 0.019698 −0.013383 −0.0210110.023642 0.045979 0.061742 −0.017575 DUS1L −0.001207 0.026681 −0.076456−0.070306 −0.051412 −0.008686 −0.006007 0.056095 0.047206 0.051313−0.006912 MYO9B −0.03253 0.000484 −0.080555 −0.029974 −0.05389 −0.010926−0.003608 0.00739 0.089177 0.036694 0.038422 ATP6V0B −0.013622 −0.016659−0.079323 −0.068862 −0.055328 −0.003415 0.016865 0.035629 0.0442220.052813 0.037998 UBE2S −0.022675 −0.034023 −0.068842 −0.078286−0.047136 0.042402 0.024117 0.01054 0.056399 0.029703 0.03158 MAP7D1−0.015088 −0.032084 −0.081105 −0.05164 −0.023898 0.008349 −0.0296840.032579 0.054235 0.078865 0.008135 TECR −0.010306 −0.040594 −0.086842−0.037739 −0.00355 −0.029611 0.044197 −0.01206 0.064649 0.0661340.007153 BAT2 0.017405 −0.034367 −0.098997 −0.042117 −0.024894 −0.0392330.031533 0.019801 0.049255 0.045196 0.035213 SMTN −0.031101 0.00673−0.003077 −0.050266 −0.050591 −0.056341 −0.020067 0.008665 0.0779530.022861 0.077155 EHBP1L1 −0.014537 0.008336 −0.053425 −0.045845−0.049305 −0.046929 −0.008121 −0.003308 0.063494 0.043723 0.080138 TPM20.019069 −0.009891 −0.044762 −0.05689 −0.04768 −0.049228 −0.021830.005019 0.068944 0.039524 0.071392 TGFB1I1 −0.01077 −0.010937 −0.05655−0.023251 −0.028333 −0.056827 −0.045286 0.028882 0.046156 0.0419790.086767 DES −0.01262 −0.037415 −0.035042 −0.054817 −0.014897 −0.067835−0.00001 0.014408 0.0856 0.041297 0.049563 UBE2M 0.018013 −0.014597−0.0431 −0.099229 −0.02696 −0.002684 0.017162 −0.023253 0.0558790.010564 0.072578 ACTG2 −0.011338 −0.082539 −0.076714 −0.018972−0.014451 0.014206 −0.005889 0.059075 0.028126 0.066333 TAGLN 0.02198−0.049739 −0.047005 −0.084739 −0.019553 −0.036552 0.020983 0.0131760.047631 0.024433 0.069044 JPH2 0.009092 −0.055863 −0.041022 −0.057461−0.008114 −0.033225 −0.011901 −0.019994 0.079378 0.033809 0.070495 ACTB−0.036755 −0.019926 −0.053364 −0.082022 0.015724 −0.03979 0.0089040.028003 0.048052 0.009568 0.076776 CNN1 0.000282 0.002222 −0.020208−0.068057 0.002252 −0.054403 0.00153 −0.043107 0.059156 0.0126170.093719 MYL9 0.045208 −0.04253 −0.034785 −0.053179 −0.033284 −0.057786−0.015614 0.004586 0.062687 0.031093 0.069379 RAB5B 0.073022 −0.04561−0.053222 −0.045552 −0.035001 −0.048972 −0.01687 0.024642 0.0400680.024152 0.057346 POLH 0.03723 −0.035859 −0.068613 −0.015304 −0.084016−0.023596 0.019943 0.011787 0.067332 0.03566 0.027223 LOC100 0.047143−0.051411 −0.051016 0.01279 −0.083205 −0.032948 −0.009438 0.0250150.036732 0.0217 0.064319 131257 TPTE2P2 0.03565 −0.053543 −0.061031−0.008301 −0.077874 −0.02919 −0.006285 0.035666 0.049489 0.0322130.051635 VWA3A 0.053442 −0.02758 −0.042213 −0.019506 −0.064595 −0.0789850.015965 0.034481 0.036602 0.029166 0.049878 CKM 0.049956 −0.0472−0.051087 −0.029799 −0.053158 −0.056843 −0.011969 0.055155 0.0418090.049388 0.025722 NDUFS8 0.071907 −0.014733 −0.085389 −0.00903 −0.043625−0.034215 −0.032259 0.034029 0.055709 0.034287 0.000658 CARM1 0.06251−0.005934 −0.031975 −0.039974 −0.011743 −0.069839 0.003849 −0.0167080.094537 −0.022286 0.03172 PTRF 0.068747 −0.047573 −0.019415 −0.072406−0.025043 −0.035028 0.008024 0.010542 0.070335 −0.027154 0.044604 SELM0.045825 0.023145 −0.018369 −0.026552 0.018006 −0.107376 −0.0382760.010143 0.056025 −0.0016 0.045511 HSPB7 0.038715 −0.01597 −0.076854−0.024481 0.017618 −0.071447 −0.013289 −0.008033 0.068451 0.0047710.06073 C1QA 0.027867 −0.009106 −0.042234 −0.048826 −0.011571 −0.056732−0.052403 0.03184 0.069425 0.073039 −0.004295 EHD2 0.050461 −0.037345−0.004609 −0.071249 −0.00558 −0.066467 −0.031757 0.028441 0.0753480.006645 0.03586 CDC42EP1 0.037986 −0.038367 0.029019 −0.062776−0.017698 −0.066136 −0.040458 0.015083 0.074027 0.049868 0.009796 PDK3−0.02938 −0.012759 −0.040263 0.001725 −0.086782 −0.047882 0.0111910.056435 0.068209 0.041058 0.013788 KILLIN −0.001368 0.037184 −0.018825−0.023218 −0.091312 −0.069872 0.018115 0.066689 0.030639 0.028380.017165 PDLIM5 0.004142 −0.041464 −0.027314 −0.050567 −0.074867−0.040075 0.019814 0.033758 0.080974 0.0294 0.033294 LOC220906 0.008516−0.030737 −0.040484 −0.020704 −0.075353 −0.027995 0.055824 0.0153190.084203 0.038038 −0.029626 VPS53 0.019727 −0.022765 −0.061814 −0.016152−0.077315 −0.03512 0.03613 0.024851 0.086561 0.025705 −0.005969 CYP20A10.031075 −0.022273 −0.050154 −0.024711 −0.085644 −0.023974 0.0375860.01647 0.081494 0.034485 −0.017331 LOC728264 0.019506 −0.01658−0.040037 −0.038179 −0.054925 −0.055633 0.017963 −0.003163 0.0923440.060204 −0.000167 PGPEP1 0.019668 −0.002645 0.011091 −0.018452−0.075644 −0.052986 −0.000902 −0.003208 0.105083 0.038364 −0.020001CCBE1 −0.002417 −0.012672 −0.012807 −0.025962 −0.073753 −0.0185410.014207 0.02453 0.091952 0.054757 −0.057162 MAP2K2 0.032207 0.03955−0.064401 −0.031648 −0.051971 −0.053757 0.048237 −0.03155 0.037850.064321 0.00889 SELO 0.01761 0.067439 −0.03322 −0.043803 −0.051404−0.060889 0.033581 −0.025993 0.067947 0.04029 −0.006219 DAPK3 0.0251330.020703 −0.013765 −0.067703 −0.055069 −0.072679 −0.00264 0.0035430.063862 0.048355 0.041567 NDUFV1 0.066694 0.006248 −0.013065 −0.050565−0.068097 −0.056277 0.000953 −0.018021 0.061292 0.054274 0.016289 ZNF3580.052209 0.012936 −0.017924 −0.017565 −0.014356 −0.069224 −0.008495−0.064191 0.089238 0.03916 0.010094 UBQLN4 0.089555 0.017245 −0.076522−0.058914 −0.004071 −0.024021 −0.004469 −0.017811 0.047098 −0.0179140.03462 ATP6V0D1 0.064547 0.020408 −0.093963 −0.039152 −0.0314980.011002 −0.001961 0.015039 0.016238 −0.046607 0.059905 KCTD13 0.121597−0.004512 −0.037559 −0.024781 −0.05055 −0.019621 −0.027925 −0.0026910.034395 −0.010581 0.021425 ATP6AP1 0.129532 −0.03464 −0.017295−0.016065 −0.031704 −0.035536 −0.032711 0.013084 0.009846 0.0099310.008489 ATP6V0C 0.091949 −0.070325 −0.042771 −0.006254 −0.05105−0.017807 0.032067 0.028159 0.023895 −0.032295 0.026181 NDUFB10 0.081475−0.061826 −0.070605 −0.022882 −0.020324 0.008416 −0.007354 0.0026280.052766 −0.035473 0.041901 CFL1 0.043681 −0.035872 −0.100005 −0.0330040.013546 −0.02343 −0.021577 0.019056 0.010281 0.010578 0.079048 FKBP8−0.066463 −0.057495 −0.067013 0.006896 −0.030761 −0.009213 0.0107250.035846 0.074157 0.044227 0.012836 BGN 0.024109 −0.071914 −0.015825−0.035829 −0.0294 −0.035763 0.027297 0.050987 0.049521 0.064071−0.054587 MMP9 −0.020196 −0.041279 −0.053242 −0.039306 −0.011605−0.026716 −0.002024 0.088116 0.011669 0.079478 −0.027405 COL5A3−0.031788 −0.074095 −0.002051 −0.005817 −0.002742 −0.020913 −0.0024380.056758 0.016406 0.094326 −0.054175 CSPG4 −0.013001 −0.077861 −0.018568−0.022911 −0.039035 −0.026343 0.007484 0.024038 0.04079 0.103051−0.008739 HLA-E −0.022565 −0.063571 −0.028781 −0.053026 −0.00332−0.005018 0.007077 0.030318 0.042952 0.097556 −0.041886 NDUFA4L2−0.048661 −0.039729 −0.019807 −0.022639 −0.041276 −0.008399 0.0195050.038664 0.000058 0.115407 −0.022198 TAGLN2 −0.012799 −0.029104−0.042057 −0.025709 −0.039083 −0.001985 −0.037224 0.051827 0.0710250.077101 −0.047745 COL6A2 −0.01639 −0.070688 −0.01671 −0.001785−0.030013 −0.012156 −0.035767 0.084238 0.054717 0.050976 −0.041812ADAMTS14 −0.037775 −0.040764 0.013578 0.019428 −0.057442 −0.005362−0.010976 0.083169 0.04647 0.038929 −0.067978 SPOCD1 −0.044388 −0.073617−0.042176 −0.014591 0.074328 0.08345 −0.021029 AEBP1 −0.010792 −0.015061−0.005414 −0.092081 −0.00016 −0.054408 0.00382 0.067828 0.0720480.019533 −0.017686 RCN3 −0.022376 0.005417 −0.00298 −0.092105 0.007117−0.03197 −0.010897 0.073631 0.050394 0.042031 −0.048693 LAPTM5 0.002226−0.027366 −0.024914 −0.081718 −0.008057 −0.02118 −0.024436 0.075050.038634 0.068693 −0.036134 LMNA −0.019008 −0.048611 −0.057604 −0.037401−0.024593 −0.006691 −0.046272 0.070111 0.079271 0.027051 0.012211 EMP3−0.037667 −0.036466 −0.055476 −0.056946 0.004379 −0.021457 −0.0352950.068855 0.07671 0.025706 0.015231 ISG15 0.040184 −0.000886 −0.045023−0.013378 −0.046076 0.004469 −0.003682 0.023697 0.089088 0.021209−0.085944 H2AFJ 0.05357 −0.017954 0.002111 −0.016142 −0.060534 −0.0813−0.036493 0.068092 0.026047 0.038931 0.020466 AFMID^(#) 0.064646−0.015807 0.003104 0.032999 −0.057733 −0.072172 −0.065185 0.0320390.018821 0.039555 0.032382 LRRN4 0.05274 −0.014181 −0.000355 0.047314−0.054286 −0.066232 −0.050819 0.073934 −0.012768 0.039152 −0.003566FASTKD2 0.007454 −0.014142 −0.013725 0.040757 −0.059579 −0.0712460.072562 −0.026472 0.074341 −0.009434 CACNA1A −0.01151 0.041553−0.018574 0.023656 −0.051353 −0.068566 −0.056757 0.058643 −0.0027890.072183 0.018214 GPM6A −0.021305 −0.004272 −0.004913 0.04437 −0.057811−0.072356 −0.037738 0.069263 −0.001981 0.068318 0.01942 C3orf14−0.016779 −0.003706 −0.024182 0.016362 −0.062357 −0.079492 −0.0198010.06059 0.010895 0.073788 0.036478 PPAPDC2^(#) −0.00193 0.02203 0.0143060.028436 −0.071638 −0.075651 −0.057939 0.056554 0.020948 0.0458130.02936 C15orf43^(#) 0.025162 0.013516 0.00852 0.00847 −0.066548−0.078711 −0.063649 0.052207 0.032986 0.043663 0.030934 CYBA 0.0403560.018431 −0.011245 −0.021374 −0.065239 −0.069087 −0.027799 0.0416540.02404 0.086234 −0.01447 CNN2 0.008112 0.007959 0.046487 −0.005186−0.074784 −0.085552 −0.025361 0.017281 0.025554 0.063263 0.038552C14orf4 −0.038153 −0.030788 −0.00609 0.016077 −0.021597 −0.033401−0.087472 0.066225 0.010342 0.066133 0.038904 PAQR8 −0.040889 −0.033509−0.003407 0.031719 −0.037434 −0.046435 −0.071122 0.059427 0.0167590.058274 0.051728 MAVS −0.039328 −0.043903 −0.042383 −0.006452 −0.038723−0.036917 −0.035742 0.078633 0.020158 0.069904 0.037206 TMEM19 −0.031429−0.052059 −0.027324 0.01745 −0.043755 −0.040997 −0.052175 0.0691450.032948 0.06095 0.042789 PPM1N −0.030279 −0.024037 −0.018718 0.030877−0.053835 −0.066875 −0.037816 0.061119 0.059677 0.006385 0.058102 LSP1−0.056907 −0.034391 −0.032368 0.003296 −0.0381 −0.050306 −0.0236570.088394 0.037702 0.021173 0.051064 SHROOM1 −0.058875 −0.038653−0.000187 0.066022 −0.035051 −0.055063 −0.036716 0.030582 0.0309410.019762 0.071424 PIAS4 −0.049085 −0.058809 −0.00818 0.038317 −0.023721−0.056601 −0.038048 0.033229 0.023693 0.045371 0.077275 C10orf57−0.017557 −0.04193 −0.039044 0.017272 −0.043753 −0.074369 −0.0194940.057816 0.0245 0.060552 0.054668 ZNF23 −0.019707 −0.047241 −0.039967−0.01502 −0.047083 −0.071379 0.009852 0.059507 0.032978 0.056498 0.05138GPR85 −0.021429 −0.031074 −0.045769 0.04329 −0.049313 −0.081114 0.0104450.034751 0.019474 0.044998 0.064705 PLA2G2D −0.022332 −0.029189−0.052854 −0.012072 −0.075907 −0.044783 0.035836 0.027995 0.0272350.042315 0.075199 NUBPL −0.016792 −0.028576 0.02022 0.067766 −0.07616−0.043675 −0.020862 0.003882 0.024089 −0.0064 0.086216 C1orf69 −0.015804−0.033378 −0.016596 0.06405 −0.07003 −0.055035 −0.010583 0.0109090.039082 0.003071 0.082884 PTPN14 0.015777 −0.049357 −0.012225 0.044635−0.091195 −0.01345 −0.031649 0.028548 0.019024 −0.00304 0.082465 SLC4A10.002913 −0.009932 0.010989 0.062478 −0.056711 −0.055473 −0.068440.028852 0.001729 0.017281 0.077907 PRKCSH −0.062073 −0.045291 0.0114180.046751 0.002572 −0.080424 −0.004167 0.036143 0.021017 0.07638−0.004248 CERCAM −0.040032 0.006234 0.031008 0.039566 −0.061133−0.055347 −0.034034 0.074875 0.024652 0.053578 −0.033175 FXYD5 −0.0416130.021635 0.054891 0.035674 −0.053841 −0.054258 −0.028148 0.0716270.012516 0.043086 −0.045259 CD248 0.001771 0.006373 0.029663 0.04942−0.014174 −0.050355 −0.061649 0.039502 −0.0136 0.088042 −0.053966 PITX1−0.056436 0.005438 −0.055993 0.048554 −0.030708 0.042447 0.046493−0.033801 0.014694 −0.057499 0.063515 PTK7 −0.019312 0.053041 −0.0640290.032325 −0.033619 0.045677 −0.062405 0.02714 0.020974 −0.0602540.045425 CRABP2 0.006334 0.002766 −0.029211 0.046143 −0.013048 0.040453−0.085078 0.000863 −0.008466 −0.052642 0.085986 NES 0.055172 −0.03304−0.025564 0.02333 −0.000431 0.021284 −0.079758 −0.067085 0.0009490.051836 0.054554 IGFBP5 0.049495 −0.006376 −0.110679 0.075644 0.0006460.001404 −0.000179 −0.012499 −0.02568 −0.007401 0.031261 PPP2R4−0.027733 −0.061253 −0.087813 0.06182 0.043959 0.02945 −0.028063−0.014893 0.005018 0.049185 0.002283 EPHB4 −0.03882 −0.02126 −0.0898540.025763 0.018591 0.020777 −0.043355 −0.01798 0.010852 0.011525 0.09157TRIOBP 0.032471 −0.001987 0.061052 −0.000476 0.016199 −0.061102−0.031404 −0.021033 −0.000151 −0.059765 0.092358 HTRA3 −0.042608−0.019144 0.005174 −0.008611 −0.005252 −0.048035 0.007813 0.053248−0.031832 −0.035981 0.11176 HMGA1 −0.003996 −0.032521 −0.010897 0.002220.030204 0.030069 0.088635 0.04648 −0.052563 −0.035852 −0.072345 SPHK1−0.005127 −0.033039 0.000594 −0.010218 −0.011469 0.044242 0.0477930.064672 0.018304 −0.029125 −0.106541 SH3BGRL3 0.003461 −0.059289−0.057422 0.024009 −0.018932 0.004944 0.018613 0.094833 0.025068−0.001634 −0.067512 SNX17 0.084863 −0.037263 −0.040309 0.00695 0.0449350.005449 −0.01847 0.064379 −0.028916 −0.060595 −0.031726 COL5A1 0.093095−0.010355 −0.022575 0.01719 0.001129 −0.028678 −0.026854 0.0673410.020878 −0.039092 −0.06848 LGALS3BP 0.066832 −0.039581 −0.0124560.075906 −0.008489 0.009964 −0.011619 0.051146 −0.019285 −0.021375−0.08131 KAT2A 0.042153 0.065113 −0.027306 −0.073878 0.019754 0.035071−0.043936 −0.04149 0.042159 0.026702 −0.048269 GSTP1 0.078373 0.020601−0.047605 −0.056568 0.044147 0.050305 −0.041367 −0.012648 0.010187−0.061813 0.002057 WFS1 0.095447 0.071108 0.016805 −0.036149 0.021226−0.00914 −0.022811 −0.042186 −0.021757 −0.055039 0.014825 LGALS1 0.075110.056662 0.013992 −0.027241 0.02392 −0.021842 −0.064256 0.02517 0.014949−0.080533 0.001346 MFAP4 0.089759 0.043849 0.010267 0.017181 −0.0018880.036643 −0.083251 −0.033221 −0.004919 −0.050234 0.001992 CLIP3 0.0265780.049827 0.052048 −0.05773 0.039048 0.029565 −0.037514 −0.0859090.018619 −0.033794 0.020153 FCGRT 0.029503 0.033488 0.087813 −0.0409060.061706 −0.002441 −0.065352 −0.023463 −0.020812 −0.041832 0.010866RNS-8S1 0.06891 0.000841 −0.035532 −0.038087 0.080127 −0.005135−0.004674 −0.047166 −0.017313 −0.055032 0.053183 GRINA 0.051917 0.0012060.02968 −0.029386 0.002505 0.060714 −0.058342 −0.038669 −0.0815820.042627 0.028407 NFATC4 0.013267 −0.04094 0.039599 −0.032066 −0.0038910.086785 −0.085478 −0.01064 0.006956 0.04321 −0.031335 CLTA 0.031155−0.056525 −0.02126 −0.062153 0.041195 0.078825 −0.064489 −0.0090080.00751 −0.018482 0.035824 CLEC11A 0.051865 −0.02857 0.061307 −0.0410660.07035 0.051123 −0.030037 −0.009186 −0.035633 −0.033101 −0.049462 VWF0.108897 −0.024999 0.048738 −0.010415 −0.016499 0.017259 0.008287−0.069874 −0.005637 −0.040542 0.012183 DPP6 0.089362 −0.081233 0.0365830.036162 0.025756 0.010694 −0.021354 −0.006062 −0.047681 −0.0378550.010255 FAM50A 0.076495 0.061743 −0.03559 −0.040981 −0.058785 0.0118330.011634 0.012511 0.027232 0.007413 −0.070842 GMPPA 0.058171 0.097579−0.037383 −0.018912 −0.013729 −0.004895 −0.006633 0.020413 0.012968−0.011285 −0.08175 PTBP1 0.099278 0.046426 −0.043869 −0.048318 0.005758−0.002759 0.034579 −0.062307 −0.027095 −0.00227 0.013564 PHPT1 0.0465370.0 0192 −0.094074 −0.041064 −0.018825 0.022702 0.036281 −0.043939−0.016944 0.07095 0.008305 SOX4 0.078473 0.012879 −0.056332 0.009347−0.071828 −0.003441 0.014124 −0.041975 0.05417 0.040496 −0.031954 BOP10.092566 0.009934 −0.08307 −0.01528 −0.059899 −0.01222 0.044118−0.010885 0.005365 0.025877 −0.003252 MBD3 0.057751 0.021609 −0.093019−0.024549 −0.01835 −0.025043 0.033418 −0.027034 0.048194 0.052612−0.036784 SSR4 0.116653 0.013962 −0.05294 −0.020422 −0.020742 0.012180.021163 0.007781 −0.049201 0.014605 −0.039608 LEPREL4 0.109761−0.034521 −0.064729 −0.030521 −0.007744 0.007843 0.011412 0.0225660.0161 0.005617 −0.053853 KRT8 0.108755 0.03639 −0.019527 0.01423−0.057146 0.01017 −0.016076 −0.038699 0.04193 −0.027711 −0.030421 HBA20.12565 0.028501 −0.016526 0.016206 −0.057809 −0.016035 −0.01944−0.029603 −0.011906 0.012873 −0.003958 HBA1 0.112095 0.015887 −0.0038830.043328 −0.053636 0.030117 −0.005791 −0.040668 −0.027883 −0.001733−0.036751 C4orf48 0.050314 0.004294 −0.043962 0.009193 −0.0145710.030787 0.090627 −0.029177 −0.052734 0.025716 −0.063861 RNF187 0.052957−0.020389 −0.036833 0.021261 0.044408 0.021605 0.05137 −0.032314−0.105054 0.000262 0.01119 TIMM13 0.099462 −0.016634 −0.043377 0.016585−0.008372 −0.022765 0.030999 −0.047482 −0.061373 0.026666 0.041288 GPAA10.099388 −0.004971 −0.005412 0.055181 0.002602 0.018272 0.020473−0.067864 −0.06102 −0.007908 −0.01357 COL4A2 0.033891 −0.084062−0.021622 −0.04982 0.011832 0.024716 0.021395 −0.070799 0.0348730.014035 0.059275 CAPNS1 0.024731 −0.073482 −0.013074 −0.109416 −0.004830.051382 0.022132 0.022947 0.020424 0.002538 0.007413 NDUFA3 0.022953−0.051092 −0.025295 −0.100277 0.016821 0.070968 0.036216 −0.033103−0.010561 0.01319 0.021623 SOX12 0.039237 −0.035554 −0.071822 −0.084439−0.002272 0.044965 0.046171 −0.033108 0.024891 −0.005465 0.036277 PXDN0.055742 −0.054427 −0.064461 −0.07018 0.001541 0.043748 0.0495380.006314 0.023375 0.01375 −0.044909 NDUFS6 0.078196 −0.0583 −0.085106−0.05082 0.006793 0.025816 0.030743 −0.025662 0.012631 0.014717 0.016758NDUFA13 0.068512 −0.077239 −0.054317 −0.027073 −0.008731 0.0502430.02796 −0.052734 0.04066 0.011041 −0.005563 NDUFB7 0.047843 −0.049855−0.067453 −0.078353 0.004045 −0.002391 0.026631 −0.029315 0.0476440.053521 0.011129 PFDN5 0.06054 −0.084885 −0.002056 −0.031126 0.0683010.045871 0.031713 −0.030604 −0.030643 −0.020657 −0.019792 MYL6B 0.011618−0.052093 −0.030343 −0.048316 0.002747 0.074794 0.090954 −0.030183−0.035097 0.002368 −0.013632 HOXD9 0.043893 −0.056688 −0.019474−0.045277 0.024864 0.07654 0.056293 −0.066834 −0.000004 −0.02383−0.007934 PABPN1 −0.037588 −0.07734 −0.022393 −0.038561 −0.0283890.07467 0.036421 0.026452 0.04213 0.023013 −0.047089 EMX2OS −0.001889−0.027209 −0.066877 −0.035046 −0.059883 0.090867 0.02461 0.0121720.046114 0.005247 −0.033421 CDC37 0.015981 −0.063723 −0.039139 −0.053305−0.011243 0.102453 0.012713 −0.032251 0.018447 0.031135 −0.022104 ERGIC30.004476 −0.080202 −0.005951 −0.03842 0.041113 0.093333 0.006186 0.011340.005492 −0.013867 −0.059649 NUCKS1 −0.000505 −0.060033 0.017572−0.062046 0.017882 0.060998 0.061061 −0.026778 −0.007824 0.043222−0.064298 NDUFS5 0.058557 −0.0209 −0.078594 −0.031123 0.036038 0.069705−0.005427 −0.039954 −0.050837 0.028387 0.011292 CRIP2 0.069322 −0.043563−0.031062 −0.014691 0.020375 0.008478 0.005087 −0.061425 0.0010740.089942 −0.045296 EDF1 0.081088 −0.067571 −0.045229 −0.039431 0.0308030.008401 0.021366 −0.052441 −0.021338 0.050453 0.018552 POLR2L 0.042244−0.026443 −0.086038 0.002587 −0.00731 0.083229 −0.019698 0.037543−0.035474 0.020544 −0.044283 NRBP1 0.046993 −0.008195 −0.077052−0.069543 0.037799 0.047663 0.001367 0.053143 −0.002341 −0.023074−0.046894 DYNC1H1 0.071374 −0.005695 −0.041937 −0.074709 0.0250460.045673 0.043205 0.016226 −0.063024 −0.031829 −0.004154 CIRBP 0.053785−0.026835 0.018776 −0.047043 0.033492 −0.013356 0.076075 −0.0892860.022397 −0.023093 0.006281 RHOT2 0.01037 0.049472 −0.010643 −0.029959−0.02771 0.047763 0.064382 0.01567 0.022942 −0.083765 −0.061351 DMWD−0.001264 0.006474 −0.044293 −0.089669 −0.032473 0.015878 0.060256−0.010871 0.083525 −0.01261 −0.008688 CDK10 0.01051 0.035535 −0.062564−0.061859 0.00115 0.052851 0.075309 −0.014289 0.026418 −0.033295−0.05217 MIF 0.042633 0.015549 −0.063422 −0.099843 0.003226 0.0459030.051107 −0.008326 0.006491 0.011849 −0.035787 PPP1R12C 0.0080090.063001 0.000513 −0.079601 −0.023439 0.017337 0.010024 −0.0649980.056603 −0.036174 0.047487 AMH 0.009967 0.04726 0.009691 −0.092835−0.003799 0.011085 0.062749 −0.06621 0.049308 −0.010045 −0.016634SNRNP70 0.020476 0.055505 0.014213 −0.050418 0.024775 0.062112 0.047358−0.063814 −0.010171 −0.06944 −0.018874 RAB11FIP3 0.020417 0.0231530.014803 −0.03973 0.021595 0.014033 0.082296 −0.04165 −0.008697−0.099048 0.022472 PTOV1 0.028338 0.041963 −0.0131 −0.097633 0.0166010.045313 0.027867 −0.040202 −0.002819 −0.059744 0.041783 RPL28 0.049572−0.026972 0.021548 −0.095035 0.023795 0.053657 0.053939 −0.0337210.004898 −0.036012 −0.028617 RPS9 0.03769 0.014788 0.006453 −0.0990850.040535 0.080235 −0.010298 −0.04513 −0.007431 −0.020362 −0.010971 COMP−0.051152 0.012708 0.053744 −0.025863 −0.079691 0.073084 0.030157−0.041778 −0.00987 0.039152 ISLR2 −0.028037 0.000836 0.05007 −0.026045−0.031495 0.070305 0.084523 −0.02323 −0.058723 −0.035664 0.001224 RPS26−0.00774 −0.070657 0.068893 0.01003 −0.013671 0.050984 0.014095−0.010777 −0.078447 −0.014574 0.054276 C9orf16 0.006893 −0.118558−0.027189 0.026392 0.003643 0.054717 0.042003 −0.01156 −0.024781−0.013084 0.032906 PPDPF −0.018669 −0.081958 −0.007497 0.063036−0.039268 0.057014 0.055646 −0.000372 −0.040052 −0.029549 0.029162 EPHX1−0.048022 0.025008 −0.042328 0.007873 0.033263 0.055148 0.057959 0.01364−0.09807 −0.01471 −0.000243 ELFN1^($) −0.069822 −0.014421 −0.002011−0.017988 0.046087 0.067854 0.068908 0.021158 −0.049327 −0.034526−0.036772 FSCN1^($) −0.085028 −0.006492 −0.015518 −0.032294 0.0400580.070627 0.037402 0.051862 −0.027198 −0.031142 −0.036954 MDGA1 −0.050432−0.025187 0.015039 0.001396 0.020606 0.07572 0.078781 0.000959 −0.023839−0.034888 −0.06896 CHST8^($) −0.039268 −0.035661 0.012645 −0.0006470.056102 0.059063 0.065535 −0.05983 −0.069485 QARS^($) −0.048774−0.019753 −0.000835 −0.060107 0.059892 0.08285 0.042287 −0.00764−0.036903 −0.047056 0.00748 CEBPA^($) −0.053093 −0.02801 0.003349−0.034417 0.066534 0.071119 0.066609 −0.013577 −0.041606 −0.033821−0.023073 TMEM132E^($) −0.068297 −0.062909 −0.025288 −0.05679 0.039620.057796 0.070111 MMP15^($) −0.020629 −0.023335 0.012252 −0.017830.051878 0.068682 0.080744 −0.051328 −0.05136 −0.03872 −0.013209AP2M1^($) −0.067238 −0.018474 0.011177 −0.034652 0.063051 0.0793770.043712 −0.040676 0.010353 −0.035607 −0.030134 MDK 0.016224 0.008116−0.001568 0.002566 0.043633 0.080574 0.02811 −0.057702 −0.046113−0.084014 0.016547 ANKRD19^($) 0.002499 −0.029404 0.007381 −0.014690.055032 0.074756 0.062964 −0.040149 −0.034947 −0.074588 −0.013896CKB^($) −0.032179 −0.017447 0.000819 −0.005388 0.032181 0.0617840.064087 0.003747 −0.046192 −0.096494 0.02289 NME3^($) −0.012932−0.011164 −0.025351 −0.016082 0.022626 0.074683 0.070759 0.001938−0.039036 −0.091825 0.007968 COX5B −0.039286 −0.020205 0.000016−0.022405 0.043441 0.098402 0.023968 −0.040552 −0.068241 0.031226−0.020921 TUBB2C^($) −0.059367 0.003366 −0.060633 −0.02482 0.0544770.093467 0.02159 −0.010199 −0.035893 0.012287 −0.027768 METRN −0.048167−0.058235 −0.050741 0.018727 0.010773 0.103778 0.029904 0.014076−0.022653 −0.001685 −0.034371 UQCR11 0.010812 −0.030711 −0.048358−0.016397 0.030606 0.042255 0.088844 −0.073854 −0.038866 −0.0076770.031709 ITPKB 0.01246 −0.039062 −0.016008 −0.037592 0.021119 0.0332420.063053 −0.080842 −0.035373 −0.005981 0.074917 RPS15^($) −0.028088−0.040939 0.019281 −0.030995 0.066746 0.035241 0.073871 −0.077312−0.034364 0.014242 −0.000484 GNB2L1 0.006644 −0.070029 0.025683 −0.032530.052305 0.060079 0.052052 −0.039716 −0.068549 −0.010322 0.014451 EEF20.035893 −0.019586 0.01714 −0.050045 0.072941 0.022838 0.03352 −0.060376−0.053487 −0.04417 0.049187 RPL36 0.045189 −0.026776 0.022336 −0.0642970.068935 −0.001189 0.04843 −0.067196 −0.044591 −0.00992 0.034921 RPL180.038156 −0.020448 0.049188 −0.078734 0.03988 0.033145 0.008577−0.055364 −0.008781 −0.057146 0.051471 UBA52 0.011001 −0.06303 −0.008295−0.039576 0.050488 0.053406 0.039683 −0.057999 0.006077 −0.0616920.048999 COX6B1 0.012373 −0.027317 −0.009084 −0.08783 0.029689 0.0714330.046826 −0.032278 −0.021927 −0.048701 0.039309 SEPW1^($) −0.007902−0.022676 −0.038083 −0.077415 0.044643 0.058774 0.049169 −0.050868−0.009189 −0.030903 0.052919 FBL 0.011847 −0.031948 0.017039 −0.0626840.072049 0.053692 0.021084 −0.057343 −0.031378 −0.046924 0.044031 RPLP1−0.010941 −0.042832 −0.006592 −0.062216 0.076227 0.056127 0.012107−0.037591 −0.037859 −0.031046 0.059382 MYL6^($) −0.001799 −0.029208−0.010295 −0.064121 0.066408 0.055153 0.026052 −0.049898 −0.022911−0.050367 0.059636 COX411^($) −0.013158 −0.026221 0.000062 −0.0732030.051986 0.07295 0.040616 −0.014331 −0.060955 −0.036731 0.033899RPS10^($) 0.002603 −0.061214 −0.01396 −0.058033 0.057874 0.072270.035696 −0.031587 −0.047711 −0.022299 0.03712 ROMO1^($) 0.000111−0.026908 −0.026995 −0.063389 0.063833 0.088708 0.026554 −0.031721−0.054508 −0.011864 0.008622 RPL4 −0.016389 −0.006778 0.056867 −0.0164450.070445 0.040377 0.038586 −0.043267 −0.071072 −0.056123 0.018403 RPL37A−0.012661 −0.006457 0.028543 −0.005708 0.067536 0.056932 0.037115−0.058372 −0.086617 −0.028396 0.019157 RPS8 −0.038372 −0.01258 0.044361−0.006462 0.07592 0.048341 0.018483 −0.044717 −0.080468 −0.0277760.031361 RPL5 −0.041306 −0.01442 0.049293 0.001127 0.072507 0.0405340.029397 −0.054852 −0.072996 −0.02903 0.031982 RPS4X −0.011388 −0.0170820.058504 0.014481 0.066147 0.056517 0.015148 −0.040763 −0.079696−0.049157 0.005467 RPL12 −0.016258 0.013968 0.060789 0.025335 0.06280.024139 0.032193 −0.061943 −0.085718 −0.027034 0.00612 RPL7A 0.0020150.019488 0.045042 0.006342 0.068745 0.029252 0.029795 −0.053796−0.089532 −0.046624 0.017505 RPS28 0.000829 −0.017981 0.059331 0.0217990.066231 0.021758 0.043487 −0.066805 −0.060909 −0.053538 0.014241 RPS19−0.01627 0.004334 0.031987 −0.044418 0.051734 0.080074 0.039189−0.070921 −0.056584 −0.012878 −0.003355 RPL14 −0.047322 0.0010360.042627 −0.03174 0.069912 0.062355 0.032145 −0.038557 −0.075346−0.01623 0.003329 RPL19 −0.044036 −0.004775 0.041728 −0.032493 0.0657710.074984 0.022054 −0.043945 −0.069079 −0.018784 0.007927 SSR2 −0.0518930.008227 0.010438 −0.0074 0.091008 0.048904 0.030051 −0.059333 −0.059484−0.022623 0.015507 FTL −0.040418 0.02324 0.046415 −0.044821 0.0843910.067121 −0.013119 −0.037577 −0.036024 −0.036995 −0.00989 RPL11−0.045627 −0.00845 0.053353 −0.011698 0.07989 0.067488 0.011725−0.019155 −0.05416 −0.048373 −0.021471 EEF1G 0.006691 −0.006993 0.037359−0.038991 0.100915 0.046985 0.017182 −0.046783 −0.048639 −0.04113−0.019229 SLC25A6 −0.00871 −0.021236 0.026419 0.008007 0.047553 0.0769110.024754 0.036643 −0.104144 −0.006728 0.000421 RPS11 −0.025565 −0.0096390.0599 −0.057346 0.059219 0.033101 0.027122 −0.058562 −0.029636−0.047837 0.052488 RPS16 −0.032842 0.006271 0.019851 −0.044538 0.0739780.0407 0.023904 −0.046316 −0.052531 −0.051335 0.059801 RPS5 0.0245430.019057 0.041498 −0.050147 0.062705 0.035646 0.035497 −0.065561−0.055935 −0.052369 0.021864 GLTSCR2 −0.000356 0.028526 0.053641−0.079196 0.067614 0.039113 0.016586 −0.050657 −0.019472 −0.0546470.006399 RPS18 −0.033774 −0.024912 0.024762 −0.060379 0.049494 0.063453−0.039447 −0.032727 −0.050245 0.018864 0.065641 PIK3R1 −0.04091 −0.008760.036847 −0.032897 0.097768 0.034334 −0.030598 −0.055594 −0.04032−0.001392 0.041594 FAU −0.043814 −0.013144 0.018366 −0.060303 0.0827960.038111 −0.018158 −0.025317 −0.042869 −0.021112 0.068443 RPS3 −0.031539−0.017711 0.066054 −0.0456 0.081799 0.043545 −0.010903 −0.035583−0.066128 0.005885 0.013477 RPLP2 0.012285 −0.020227 0.012363 −0.0404670.085547 0.053262 −0.011986 −0.045866 −0.057608 −0.042109 0.049673RPL27A −0.004616 −0.016177 0.014554 −0.041499 0.089512 0.073616−0.026676 −0.021308 −0.067092 −0.027123 0.016159 RPS2 −0.076057−0.013095 0.026145 −0.041661 0.05017 0.070365 0.033093 −0.002571−0.068421 −0.016523 0.020311 RPL15 −0.072712 −0.011628 0.030178−0.004565 0.069047 0.056676 −0.012165 −0.019745 −0.079438 0.0059990.033086 RPS24 0.003513 0.002953 0.036513 −0.005668 0.062752 0.0016950.068575 −0.046949 −0.06747 −0.06681 0.032599 SRRM2 −0.079763 0.0060210.025255 −0.00865 0.050756 0.042795 0.033753 −0.006677 −0.043656−0.073731 0.047622 LOC404266 0.037659 0.01685 0.098762 −0.086961−0.057395 H1FX −0.065508 −0.063956 0.009728 0.024471 0.048738 0.0485390.070866 −0.05634 −0.010961 −0.000974 −0.013732 RNASEK −0.003495−0.054669 −0.050941 −0.0007 −0.004544 0.011885 −0.010341 0.007263−0.069379 0.056228 0.091756 KDM6B −0.060514 0.001105 −0.0097 −0.013307−0.036954 0.024749 0.025377 −0.022592 −0.06641 0.063395 0.082561 SERF2−0.09308 −0.028009 −0.025765 −0.02724 0.052805 0.021341 0.00347 0.0670810.035698 0.000577 −0.049273 GNAI2 −0.088531 −0.058851 −0.023766−0.029188 0.016712 0.032717 −0.011014 0.07415 0.028417 0.033608−0.026231 S100A11 −0.090543 −0.039576 −0.032343 0.023918 0.0281350.044856 −0.04627 0.052559 0.010018 0.045185 −0.032877 CST3 −0.055032−0.08132 −0.009021 0.01649 0.004805 0.032859 −0.015388 0.095609 −0.0396−0.003984 0.015905 COL6A1 −0.069779 −0.063088 −0.024438 0.0150190.024943 0.024161 −0.022869 0.095093 −0.032144 0.026083 −0.01246FOXP4^($) −0.008059 −0.061332 −0.071757 −0.041369 0.02723 0.0353160.04645 0.00548 0.000058 −0.049506 0.070697 MAPKAPK2 −0.05859 −0.003595−0.076834 −0.022338 0.016595 −0.016445 −0.011089 0.069557 −0.0367820.067586 0.032193 EIF5A −0.060294 −0.033486 −0.066135 −0.034717−0.012491 0.012254 0.003992 0.034099 −0.025174 0.086092 0.049459GNB2^($) −0.091733 −0.04987 −0.055687 −0.038417 −0.001269 0.0300690.009392 0.049407 0.02588 0.012215 0.050787 PTMS −0.092155 −0.019997−0.07792 −0.011353 −0.004888 0.050627 −0.021819 0.02933 0.0185990.037829 0.038976 GAPDH^($) −0.070979 −0.070644 −0.062876 −0.0233140.009056 0.041773 0.02077 0.048178 −0.011496 0.054821 0.008363 EIF4A1−0.048745 −0.021661 −0.070264 −0.067521 0.00079 0.060356 −0.0015310.050274 −0.032199 0.042334 0.031125 BCYRN1^($) −0.022577 −0.052181−0.090681 −0.042753 0.031596 0.019523 0.047645 0.066994 0.006929 0.00797−0.027922 ALDOA^($) −0.055463 −0.022221 −0.094021 −0.059792 0.0083130.033223 0.041551 0.051918 0.01466 0.022737 −0.000831 PFN1 −0.004153−0.063736 −0.085149 −0.057557 −0.015657 0.009314 0.007251 0.0297540.018942 0.033981 0.069904 RALY −0.018768 −0.08998 −0.079905 −0.0405640.01216 0.023154 −0.007811 0.040406 0.043125 0.021733 0.033522 TUBB^($)−0.009644 −0.078529 −0.067406 −0.073258 0.035844 0.056806 0.0080460.025901 0.003334 0.008989 0.028339 MIDN^($) −0.072614 −0.026722−0.062457 −0.036645 0.029949 0.019687 0.090841 −0.021605 −0.0000880.030199 0.012896 BSG −0.079026 −0.051983 −0.020591 −0.023936 0.065276−0.013637 0.083124 −0.022668 0.010048 0.015487 0.012525 MRPS21^($)−0.089706 −0.048083 −0.017406 −0.038892 0.055643 0.064821 0.003249−0.011306 −0.023674 0.036037 0.029022 CALM3 −0.076628 −0.034812 0.011408−0.065789 0.069671 0.062071 0.003938 −0.014872 −0.021751 −0.0031750.036656 RPL29 −0.112685 −0.03681 0.004895 −0.027467 0.027024 0.0494330.040839 −0.027472 −0.006489 0.035887 0.023553 FAM20C^($) −0.073279−0.031559 −0.012932 −0.058144 0.058913 0.044207 0.039228 0.041886−0.029534 0.02804 −0.044963 GUK1 −0.009693 −0.05822 −0.061248 −0.0247060.002744 0.038447 0.036136 0.022694 −0.057101 0.087743 −0.014185TIMP1^($) −0.01103 −0.08466 −0.046989 −0.058199 0.040801 0.0276180.050347 0.04493 −0.04304 0.022828 0.008599 TPI1^($) −0.014242 −0.061763−0.065641 −0.037993 0.045221 0.064705 0.042254 0.009472 −0.0556970.036058 −0.005797 LSMD1^($) −0.022042 −0.078965 −0.032465 −0.0182890.054373 0.070804 0.050198 −0.030522 −0.048709 0.019644 0.004806CCND1^($) −0.051348 −0.084964 −0.045745 −0.03034 0.040859 0.0472990.054694 −0.004065 −0.028674 0.021065 0.035543 SPTBN1 −0.022102 −0.08351−0.009123 −0.036312 0.064821 0.044363 0.015304 −0.036505 −0.0477490.036947 0.045459 RPL35 0.022079 −0.068549 −0.02841 −0.070487 0.032490.045095 0.028467 −0.050986 −0.016396 0.067088 0.00904 C12orf57^($)−0.028884 −0.049578 −0.014856 −0.055952 0.047335 0.03716 0.02375−0.037167 −0.040502 0.091984 0.000606 TBX2 −0.01034 −0.073855 −0.016424−0.077411 0.05548 −0.009304 0.046227 −0.003231 −0.023373 0.0669840.011991 SOD3 0.013577 −0.084261 −0.015594 −0.084598 0.044872 −0.034230.00156 0.05 0.033899 0.00393 0.027855 ACTN4 −0.035039 −0.055338 0.00523−0.093696 −0.018603 0.014718 0.01493 0.020965 0.087851 −0.0032360.016209 FLNB −0.020123 −0.018631 −0.024207 −0.061688 0.023478 0.026716−0.020765 −0.018099 0.0962 −0.062307 0.044817 TRIM28 −0.08812 0.04033−0.053186 −0.066223 −0.011958 0.042967 0.011588 0.014015 0.031419−0.010767 0.047787 SLC4A2 −0.027173 0.07888 −0.084222 −0.057904−0.031732 0.008886 0.011636 0.026367 0.037435 −0.023628 0.032087 EIF3B−0.034251 0.061592 −0.044774 −0.110025 −0.013446 −0.000202 0.0184240.025079 0.024118 0.001888 0.038743 KDELR1 −0.006466 0.01062 −0.02103−0.110642 0.013823 0.038496 −0.053316 0.064984 0.02006 −0.0132120.012583 FOSB −0.001671 0.100432 0.01034 −0.095754 0.021914 −0.01835−0.022952 −0.010893 −0.020476 0.015049 0.02897 CACNB3 −0.006471 0.105140.032421 0.015093 0.00009 0.027269 −0.021248 −0.087292 −0.018675−0.022016 0.016995 SPSB3 −0.015623 0.116962 −0.010641 −0.014906 0.0030760.032568 0.004147 −0.03133 −0.009858 −0.074681 0.017591 CPSF3L −0.0005880.111476 0.005749 −0.035772 0.013707 0.039593 0.004136 −0.028559−0.027374 −0.071921 0.007898 TSPYL2 −0.029583 0.12125 0.021609 −0.0062510.011512 0.00459 −0.033826 −0.012286 0.013424 −0.067779 0.002621 NUMA1−0.059885 0.101871 −0.003767 0.01716 0.007256 0.021371 −0.049497−0.023614 0.025748 −0.05423 0.030317 RGS12 0.039494 0.12855 −0.0152140.016595 −0.045559 −0.028371 −0.007463 −0.005726 −0.019711 −0.007899−0.017341 TMEM120B −0.004058 0.112827 −0.042763 0.048673 −0.037323−0.020861 −0.032607 −0.0102 −0.026246 −0.003014 0.040927 NR2F6 −0.0409560.122629 −0.054259 −0.008144 0.015057 0.001995 −0.011595 −0.039729−0.000067 −0.001555 0.025318 AKNA −0.044853 0.131556 −0.010093 −0.007163−0.008636 −0.038137 −0.018849 −0.000942 −0.017257 0.019024 0.017193 CAD−0.033675 0.107677 −0.062898 0.000618 −0.0113 0.01761 0.000423 0.052424−0.010562 −0.022732 −0.04258 TMEM214 0.009892 0.109627 −0.019546−0.000267 0.027958 0.017899 0.009116 0.016918 −0.029672 −0.064041−0.057977 TSKU −0.015901 0.07638 −0.018121 0.051546 0.015521 −0.020767−0.077722 0.026519 −0.060582 −0.003487 0.046278 NAB2 −0.080626 0.066823−0.02002 0.002656 0.033831 0.043336 −0.041688 0.000095 −0.05148−0.016193 0.057148 NISCH −0.08417 0.098175 −0.006354 −0.02863 0.0144560.045286 0.012959 −0.029022 0.01707 −0.005694 −0.033701 U2AF2 −0.1159970.057403 −0.04642 0.014905 −0.012775 0.044482 0.002829 0.020667 0.023178−0.008032 −0.002529 MXD4 −0.084513 0.048798 −0.051664 −0.027492−0.004867 0.083675 −0.0184 0.020965 −0.01504 0.03253 −0.016616 AUP1−0.051959 0.078841 −0.035843 −0.046816 0.000389 0.016995 −0.0099090.053855 0.007131 0.040248 −0.070442 TMED9 −0.071575 0.027233 −0.0550670.036768 0.030981 0.026725 −0.051262 0.075069 −0.028618 0.018885−0.030834 CTSA −0.089829 0.035824 −0.016388 0.035085 −0.008524 0.036795−0.015787 0.073604 0.013691 −0.017236 −0.06231 SEC61A1 −0.0316340.023904 −0.055246 0.036141 0.000503 0.044145 −0.033207 0.0336660.024233 0.042043 −0.099206 MVP −0.085514 0.019431 −0.017381 −0.0380560.008944 −0.011229 −0.021193 0.078609 0.069433 −0.001447 −0.034844TMSB10 −0.026675 0.009737 −0.063662 −0.005504 −0.052325 0.069715 0.059190.036475 −0.040559 0.033352 −0.047258 ADAMTS10 −0.042156 0.0469030.002419 −0.009516 −0.031613 0.026682 0.024834 −0.06229 0.101486−0.015175 −0.038745 FAM113A 0.054511 0.04022 0.044171 −0.013249−0.001153 0.026881 0.014441 −0.043531 −0.02293 0.037261 −0.104187 IGFBP20.043129 0.013755 0.043155 −0.004216 0.00579 0.074182 −0.005418−0.022926 −0.03663 0.012147 −0.103933 GP1BB 0.064057 0.056532 0.014911−0.022611 0.021644 0.012556 0.019629 −0.077843 0.009043 0.016695−0.082753 ATHL1 0.058005 0.06356 0.051361 0.013741 0.001216 −0.024604−0.017502 −0.005606 −0.033215 0.033512 −0.094395 EEF1A2 0.0269980.015702 0.062168 0.054365 −0.016733 0.005048 0.029611 −0.024183−0.112914 DPP7^(#) 0.059126 0.059706 0.052737 0.056367 −0.004302−0.009565 −0.001068 −0.029615 −0.022648 −0.018463 −0.08632 CPZ 0.0321340.056398 0.061638 0.059764 0.022847 −0.016148 0.002698 −0.030305−0.01338 −0.031582 −0.088562 RASSF7 0.052528 0.044697 0.047523 0.018721−0.003447 0.017498 0.0599 −0.040484 −0.022605 −0.05757 −0.074673 C1QL10.019423 0.021569 0.043947 0.050987 0.013149 0.024943 0.008428 −0.0042420.024885 −0.072524 −0.100875 WBP1 0.003734 0.030443 −0.018809 −0.0207250.052095 0.077879 0.025181 −0.010417 −0.043434 −0.002746 −0.095774 GPC1−0.000705 0.026913 0.016693 0.032024 0.005509 0.053607 0.037912 0.011206−0.038127 −0.008947 −0.11951 MMP17 −0.003341 0.026227 0.005274 0.009190.024024 0.048413 0.05765 0.025633 −0.018785 −0.079452 −0.089074 OBSL1−0.004724 0.018212 0.018152 −0.003727 0.030723 0.085028 0.059467−0.055948 −0.030561 −0.062623 −0.043809 EMID2 0.019471 0.01786 0.0602210.026046 0.040304 0.056326 0.026739 −0.040305 −0.070044 −0.04999−0.052609 LMF1 0.021086 −0.002298 0.019283 0.016993 0.042498 0.0445550.066795 −0.059846 −0.05293 −0.081031 NTNG2 −0.004604 −0.027888 0.0272320.008193 0.037094 0.066171 0.070334 −0.040827 −0.063469 −0.065635 FGFR30.007385 0.028298 0.032045 0.033481 0.02719 0.041408 0.054049 −0.014083−0.021139 −0.084006 −0.078942 BAI1 0.00266 0.02665 0.022013 0.0259450.026968 0.048367 0.057155 −0.037398 −0.04353 −0.103974 SOX8 −0.0079010.014771 0.026043 0.026561 0.060982 0.042895 0.051162 −0.063715−0.073497 −0.054867 KIF1A 0.001612 0.033211 0.032139 0.034307 0.0327990.050712 0.058894 −0.04366 −0.045731 −0.070594 −0.05385 ECEL1 −0.006993−0.02362 0.029198 0.044981 0.026551 0.050737 0.049423 −0.090677−0.069897 HAGHL 0.007178 0.005335 0.011137 0.01447 −0.006824 0.0511220.082929 −0.043055 0.024724 −0.085167 −0.051414 SLC25A29 0.0117760.016704 −0.007721 −0.033107 −0.002465 0.078957 0.088233 −0.025097−0.042875 −0.028667 −0.058512 LTBP4^(#) 0.019236 0.066143 0.0700470.031384 −0.026616 −0.060797 −0.045206 −0.01838 0.011616 −0.0489390.048578 BTBD2 −0.005384 0.057351 0.023007 0.093921 −0.007403 −0.083807−0.012108 −0.0014 −0.000766 −0.042147 0.025293 TMEM132A −0.0146020.04126 0.063091 0.069509 −0.056187 −0.032695 −0.001738 −0.034918−0.05007 0.002577 0.060191 C10orf116 −0.013898 0.027647 0.07189 0.092143−0.040855 −0.018875 0.008702 −0.068829 −0.023555 0.002411 0.017236BMP1^(#) 0.053006 0.088706 0.02398 0.015987 −0.026397 −0.05959 −0.066142−0.000239 0.024804 0.020062 −0.034103 MAP1LC3A^(#) 0.056674 0.0129330.074347 0.05924 −0.006663 −0.063652 −0.040892 −0.043472 −0.0081890.039763 −0.024292 PODXL2 0.019636 0.009176 0.062625 0.093529 −0.017248−0.051196 −0.019306 −0.020947 −0.041987 0.051046 −0.032778 AMN^(#)0.041533 0.048098 0.077435 0.076346 −0.03885 −0.033302 −0.007622−0.026063 −0.030761 −0.046337 SEMA6B^(#) 0.039827 0.036421 0.0631410.09563 −0.046745 −0.030824 −0.010896 −0.039689 0.003707 −0.019335−0.032421 CALY^(#) 0.036646 0.037142 0.084463 0.081888 −0.033437−0.017402 −0.029605 −0.045155 −0.028738 −0.024252 SPINT1^(#) 0.0411960.04696 0.061495 0.076009 −0.067195 −0.035619 −0.047897 0.00991−0.022865 −0.012329 GCGR^(#) 0.041008 0.062137 0.059836 0.047377−0.085892 −0.026405 −0.053574 ECM1 −0.002382 0.084621 0.044664 0.083265−0.013476 −0.018564 −0.032906 −0.015373 −0.001267 −0.011003 −0.065193IFITM1^(#) 0.011676 0.066801 0.042544 0.089706 −0.010816 −0.048772−0.044228 −0.032424 0.015142 0.010489 −0.044879 LY6E 0.024702 0.0495620.04419 0.083282 0.001452 −0.019376 −0.068503 0.004697 −0.0038660.001977 −0.072429 MZT2B^(#) 0.063394 0.040425 0.023325 0.080566−0.02313 −0.012738 −0.041183 −0.065564 0.006263 0.026185 −0.046986RARRES2^(#) 0.012912 0.041136 0.064012 0.046658 −0.059067 −0.049364−0.014429 −0.069137 0.041369 0.039082 −0.005132 MMP2 0.006212 0.0291020.066127 0.024273 0.046519 0.004148 −0.039784 0.037434 −0.012867−0.099058 −0.036353 ISLR 0.024912 0.034929 0.065805 0.033256 0.044915−0.008237 −0.017183 0.010498 −0.043231 −0.103228 −0.004466 HTRA1−0.029809 −0.0205 0.025668 0.012807 0.067906 0.056931 −0.042121 0.03517−0.008296 −0.095451 −0.011722 IFITM3 −0.036797 0.047354 0.0448320.080272 0.044228 −0.029378 −0.081745 −0.012587 0.006714 −0.001631−0.023555 ASS1 −0.013377 0.059631 0.040231 0.035861 0.022082 0.013186−0.112287 0.004034 0.027707 −0.035762 −0.018452 SEMA3B −0.0301860.068094 0.066948 0.037715 0.024645 0.001501 −0.057048 0.016007 0.015859−0.040366 −0.069704 ADAMTSL4 −0.019876 0.104185 0.047605 0.0354630.029562 −0.055139 −0.04819 −0.004185 0.004104 −0.014921 −0.032334 NBL1−0.046132 0.035405 0.006867 0.100647 0.04167 −0.003004 −0.047172−0.009784 −0.013339 −0.06289 0.023543 ITM2B −0.016962 0.043076 0.036450.112195 0.001892 −0.010601 −0.059387 −0.00792 −0.049245 −0.010090.004513 FBLN1 −0.035932 0.05587 0.087056 0.045262 0.054232 −0.016244−0.030864 −0.003746 −0.036077 −0.040117 −0.034494 PHLDB1 −0.0501320.058767 0.049353 0.079966 0.041396 −0.032159 −0.012172 −0.001798−0.064723 −0.020235 −0.004197 COL3A1 −0.025593 −0.018997 0.0547950.087217 0.011496 −0.024787 −0.08184 0.040681 0.021119 −0.012071−0.031561 TSPAN4 0.016146 −0.025511 0.101925 0.070223 −0.002095−0.019239 −0.067923 −0.004897 −0.004008 −0.03181 0.006401 TMEM119−0.023416 0.02713 0.009928 0.074504 0.040341 0.02667 −0.051501 0.054891−0.032795 −0.049668 −0.061906 MRC2 −0.033092 0.012677 0.02902 0.0314080.053161 0.047617 −0.081174 0.055154 −0.049512 −0.025415 −0.037496IFITM2 −0.011216 −0.007388 0.063234 0.042392 0.074193 0.022289 −0.0450070.025091 −0.057811 −0.021408 −0.062284 PCOLCE −0.03314 0.001445 0.0346810.021606 0.040465 0.025633 −0.059822 0.065605 −0.014391 0.01324−0.094777 PLOD1 0.044203 0.043214 0.013245 0.037819 0.014729 0.004141−0.087427 0.042192 0.011887 −0.022188 −0.081203 HSPB6 0.013493 −0.0216570.022173 −0.028463 0.093566 −0.029346 −0.027916 −0.058827 −0.002277−0.029174 0.075149 C19orf56 −0.048486 0.021386 0.007554 0.0112780.111747 −0.009865 −0.022847 −0.051092 −0.006797 −0.043288 0.041019 ABL1−0.072789 −0.000532 0.059016 0.037625 0.043461 0.017894 −0.034552−0.018777 −0.029997 −0.055033 0.066569 EEF1A1 −0.030069 −0.01458 0.059630.028261 0.030948 0.003903 −0.048818 −0.006135 −0.039743 −0.0606840.090473 CDC42EP4 −0.018065 0.004749 0.024598 0.026016 0.053578 0.050002−0.031444 −0.033871 −0.033505 −0.092239 0.059533 RPL26 −0.022369−0.023692 0.100335 0.039285 0.045518 0.020782 −0.008362 −0.022552−0.066956 −0.043722 0.012772 CHCHD10 −0.029824 0.026594 0.0562570.023011 0.086731 0.003728 −0.037999 −0.016245 −0.084659 −0.0173650.017566 CD81 −0.035628 0.018035 0.061008 0.037107 0.069646 0.031955−0.047636 −0.056748 −0.054877 −0.021888 0.027697 FTH1 −0.028243 0.0160480.055453 0.033826 0.086966 0.005719 0.002635 −0.044255 −0.036046−0.077179 0.01486 VWA1 0.026505 −0.04176 0.050636 0.060954 0.0577490.011123 −0.037536 −0.058587 −0.042083 −0.041047 0.042787 RPS25−0.069841 −0.014072 0.053188 −0.006269 0.085035 −0.006965 −0.029553−0.011309 −0.057396 0.009561 0.051981 DCHS1 0.00015 0.036004 0.0019890.0428 0.035732 0.010731 −0.04148 −0.050276 0.003125 −0.091162 0.072248PSD 0.040071 0.03039 −0.020637 0.02704 0.018496 0.01486 −0.01861−0.041875 −0.035154 −0.085253 0.086367 SFRP1 0.008535 0.04835 −0.0039220.071481 0.015535 −0.016026 −0.014177 −0.072868 −0.069446 0.062812 MMP110.005486 0.078499 0.008836 0.063918 0.041397 −0.014806 −0.018089−0.037626 −0.066648 −0.051624 0.03426 TBX1 0.039112 0.099958 0.0434740.03136 0.020951 −0.017925 −0.00933 −0.028694 −0.037142 −0.066346−0.021582 SNED1 −0.004265 0.086462 0.042613 0.04766 0.038901 0.0189440.000477 −0.039359 −0.035822 −0.045909 −0.0626 SERPINF1 0.04849 0.053975−0.013889 0.074365 0.032749 −0.036186 0.007012 −0.070178 −0.061609−0.013151 SAT2 0.046598 0.058946 0.054384 0.016623 0.020769 0.036659−0.006958 −0.047 −0.085025 −0.046377 −0.004441 KRT19 0.08804 0.0724170.044586 0.023113 −0.028529 −0.024335 −0.031167 −0.045158 −0.048769PTH1R 0.027273 0.048457 0.035574 0.004628 0.05182 0.071586 −0.03727−0.043059 −0.066146 −0.045271 −0.02135 CACNA1G 0.005892 0.0487890.015325 0.025182 0.041902 0.066201 0.000441 0.005037 −0.065217−0.082422 −0.042635 ANGPTL4 0.062565 −0.008966 0.093289 0.048635−0.004322 0.019516 −0.036674 −0.061426 −0.036813 −0.015708 −0.011395RPS27 0.00404 0.030714 0.085214 0.072535 0.032565 −0.023207 −0.014098−0.045881 −0.038726 −0.057627 0.010318 IGFBP4 0.003956 0.049615 0.0310910.085416 0.053591 −0.033291 −0.032252 −0.047524 −0.032966 −0.0516440.023227 PTGDS 0.009783 0.004564 0.073692 0.069126 0.042062 0.009275−0.034069 −0.090389 −0.009871 −0.02652 −0.000117 RBP1 0.018348 0.0495210.066169 0.061858 0.055548 −0.00137 −0.045467 −0.038811 −0.053989−0.03065 −0.028344 CRLF1 0.030842 0.017853 0.056271 0.050882 0.0561460.029376 0.005894 −0.078554 −0.046178 −0.038245 −0.038356 NPDC1 0.0163590.050609 0.055156 0.044605 0.04041 0.033512 −0.011126 −0.095109−0.025962 −0.034981 −0.02507 TMEM59L 0.035245 0.027255 0.071597 0.0621930.035157 0.023163 −0.020722 −0.050958 −0.042913 −0.063139 −0.026581LAMC3 0.000685 0.009951 0.082964 0.075159 0.025804 −0.003694 −0.029783−0.077873 0.00755 −0.044646 PCSK1N 0.026409 0.020207 0.059495 0.076881−0.000782 0.006883 −0.037242 −0.072629 −0.060985 −0.004245 0.036182GABARAP 0.053294 0.01325 0.050356 0.039338 0.011941 −0.006516 0.037251−0.052314 −0.086336 −0.048421 0.033247 NPW 0.041469 0.035391 0.0602160.058683 −0.016375 0.034371 0.024491 −0.033738 −0.073476 −0.05237−0.030683 RAMP1 0.033963 0.033888 0.028154 0.044487 0.036322 −0.020792−0.02024 −0.114807 0.007372 −0.020936 0.037472 SHC2 0.061167 0.0461480.058129 0.017879 −0.024096 −0.019536 0.034242 −0.096547 −0.0348180.00559 0.006566 PRDX2 0.005661 0.015533 0.015424 0.050532 −0.0171450.016709 0.039847 −0.123283 −0.017082 0.008714 0.038991 NGFR −0.0332620.006737 0.006113 0.049982 0.033394 0.051332 0.045643 −0.094842−0.058632 0.018571 −0.003737 CTXN1 0.012659 0.008398 −0.001684 0.012879−0.004593 0.022663 0.052676 −0.100538 0.028213 −0.06956 0.053489

TABLE 5 Specificity of YWHAE-FAM22A/B genetic rearrangement by FISHassays in uterine and extrauterine mesenchymal tumors (n = 827 cases,representing 55 tumor types) FISH screen for YWHAE-FAM22A No. of casesNo. of positive and YWHAE-FAM22B screened cases Uterine lesions ClassicESS 38 0 Uterine adenosarcoma/carcinosarcoma 16 0 Uterine leiomyosarcoma105 0 Uterine leiomyoma 66 0 Polypoid endometriosis 7 0 Soft-tissuetumors Leiomyosarcoma 206 0 Undifferentiated pleomorphic sarcoma 59 0Gastrointestinal stromal tumor 51 0 Desmoid type fibromatosis 22 0Angiosarcoma 21 0 Solitary fibrous tumor 13 0 Dedifferentiatedliposarcoma 12 0 Embryonal rhabdomyosarcoma 12 0 Synovial sarcoma 12 0Dermatofibrosarcoma protuberans 10 0 Myxoid liposarcoma 10 0 Malignantperipheral nerve sheath tumor 7 0 Myxofibrosarcoma 6 0 Other benign andmalignant 154 0 mesenchymal tumors Total 827 0

In the work reported herein, the inventors have identified an oncogenicmechanism for 14-3-3 proteins Ian the form of a transformingYWHAE-FAM22A/B fusion oncoprotein. The translocation-mediatedYWHAE-FAM22A/B, fusions define a previously unrecognized group ofuterine sarcoma, which is clinically more aggressive and histologicallyhigher grade than JAZF1-rarranged ESS. YWHAE-FAM22A/B oncogenic fusionresults in nuclear accumulation of the functionally intact YWHAEprotein-interaction domain. Known cytoplasmic YWHAE protein-proteininteractions are thereby likely redirected to the nuclear compartment.Disruption of YWHAE interaction in the nuclear compartment thereforewould appear to be a rational therapeutic approach. This unique geneticfusion provides a compelling opportunity to characterize 14-3-3functions in cancer development and progression.

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(Nucleotide sequence of nucleic acid encoding YWHAE-FAM22A) SEQ ID NO: 1ATGGATGATCGAGAGGATCTGGTGTACCAGGCGAAGCTGGCCGAGCAGGCTGAGCGATACGACGAAATGGTGGAGTCAATGAAGAAAGTAGCAGGGATGGATGTGGAGCTGACAGTTGAAGAAAGAAACCTCCTATCTGTTGCATATAAGAATGTGATTGGAGCTAGAAGAGCCTCCTGGAGAATAATCAGCAGCATTGAACAGAAAGAAGAAAACAAGGGAGGAGAAGACAAGCTAAAAATGATTCGGGAATATCGGCAAATGGTTGAGACTGAGCTAAAGTTAATCTGTTGTGACATTCTGGATGTACTGGACAAACACCTCATTCCAGCAGCTAACACTGGCGAGTCCAAGGTTTTCTATTATAAAATGAAAGGGGACTACCACAGGTATCTGGCAGAATTTGCCACAGGAAACGACAGGAAGGAGGCTGCGGAGAACAGCCTAGTGGCTTATAAAGCTGCTAGTGATATTGCAATGACAGAACTTCCACCAACGCATCCTATTCGCTTAGGTCTTGCTCTCAATTTTTCCGTATTCTACTACGAAATTCTTAATTCCCCTGACCGTGCCTGCAGGTTGGCAAAAGCAGCTTTTGATGATGCAATTGCAGAACTGGATACGCTGAGTGAAGAAAGCTATAAGGACTCTACACTTATCATGCAGTTGTTACGTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAACCCTGGCACCTCCCTGTCTGTGTTCACGGCTCTGCCCTTCACCACACCCGCTCCCGGCCCAGCACACGGGCCGCTCCTTGTGACTGCAGGGGCTCCTCCAGGCGGCCCTCTGGTGCTGTCTACCCTCCCCAGCACACCTCTGGTGACAGAACAGGATGGCTGCGGCCCGAGTGGGGCCGGGGCTTCCAACGTCTTTGTCCAGATGAGGACAGAGGTGGGGCCTGTGAAGGCCGCTCAGGCGCAGACCTTGGTCCTAACTCAGGCCCCCCTCGTCTGGCAGGCTCCAGGCGCCCTCTGCGGAGGTGTTGTGTGTCCACCTCCCCTACTCCTGGCAGCTGCTCCTGTGGTGCCTGTTATGGCTGCCCAGGTGGTTGGGGGCACCCAGGCCTGTGAGGGAGGCTGGTCCCAGGGCCTTCCTCTTCCACCACCACCACCACCGGCTGCCCAGCTGCCCCCCATTGTGTCCCAAGGGAATGCTGGGCCATGGCCACAAGGGGCTCACGGAGAGGGCAGCCTGGCTTCCTCCCAGGCCAAGGCCCCGCCAGATGACTCCTGTAACCCCAGGAGTGTCTATGAGAACTTCCGACTCTGGCAGCACTACAAGCCCCTGGCCCGGAGGCACCTTCCCCAGAGTCCTGACACCGAAGCGCTTTCGTGCTTCCTCATCCCAGTTCTCCGATCCCTGGCCCGGCGGAAGCCCACCATGACCCTGGAGGAGGGACTGTGGCGGGCCATGCGGGAATGGCAGCACACGAGCAACTTTGACCGGATGATCTTCTACGAGATGGCGGAAAAGTTCCTGGAGTTTGAGGCTGAGGAGGAGATGCAGATTCAGAAATCGCAATGGATGAAGGGGCCCCAGTGCCTGCCTCCTCCAGCCACACCGAGGCTTGAACCTCGAGGACCCCCGGCCCCTGAGGTGGTCAAGCAGCCAGTGTACCTTCCCAGCAAGGCCGGCCCCAAGGCCCCGACTGCCTGCCTGCCACCACCCAGGCCCCAGAGGCCAGTGACCAAGGCCCGCCGGCCACCACCCCGGCCCCACCGGCGAGCAGAGACCAAGGCCCGCCTGCCACCACCCAGGCCCCAGAGACCAGCAGAGACCAAGGTCCCTGAGGAGATCCCCCCAGAAGTGGTGCAGGAGTATGTGGACATCATGGAGGAGCTGCTGGGGCCTTCCCTCGGGGCCACGGGGGAGCCCGAGAAACAACGGGAAGAGGGCGAAGTGAAGCAGCCACAGGAAGAGGACTGGACGCCCCCAGACCCGGGCCTCCTGAGCTACACTGACAAGCTGTGTTCCCAGAAAGACTTCGTCACCAAGGTGGAGGCCGTCATTCATCCCCAATTCCTGGAAGAATTGCTTTCCCCAGATCCACAGATGGATTTCTTGGCCCTAAGCCAGGAGCTGGAGCAGGAGGAAGGACTCACCCTTGCCCAGCTAGTGGAGAAGCGCCTCCTACCCTTGAAGGAGAAACAGCATGCGAGGGCAGCCCCTAGTCGTGGCACAGCCCGGTTGGACTCAAGTTCTTCTAAGTTTGCAGCTGGCCAAGGAGCAGAGAGAGACGTCCCTGTCCCCCAACAAGGGGTTGGCATGGAAACCTGCCCACCCCAGACGACTGCCCGGGACTCTCAGGGACGAGGCAGAGCACACACTGGCATGGCCAGGTCCAAAGACTCTGTTGTGCTTTTGGGATGTCAGGATTCCCCTGGGCTGAGGGCTGCCCGGCCAACCTCTCCTCCCCAGGACCACAGACCCACCTGCCCTGGCGTGGGTACCAAGGATGCCTTGGATCTCCCTGGAGGGTCTCCTGTCAGGGAGTCACATGGGCTGGCTCAGGGGTCAAGTGAGGAGGAGGAACTCCCCAGCCTGGCCTTCCTCTTGGGTTCCCAGCACAAGCTTCTGCCCTGGTGGCTACCCCAGAGCCCTGTCCCTGCCTCGGGCCTTCTCAGCCCAGAAAAGTGGGGACCCCAGGGAACTCATCAGTTCCCATCTGCTGAGAGAAGAGGCCTCAACCTAGCACCTTCTCCTGCCAACAAGGCCAAGAAGCGACCTCTCTTTGGAAGCCTGTCCCCTGCTGAAAAGACACCCCACCCAGGGCCTGGGCTCAGGGTCTCTGGGGAGCAATCCCTGACTTGGGGGCTGGGTGGCCCCTCACAGTCTCAAAAGAGAAAGGGTGACCCCTTGGTCTCCAGGAAGGAGAAGAAGCAGCGTTGTAGCCAGT AG(Nucleotide sequence of nucleic acid encoding YWHAE-FAM22B) SEQ ID NO: 2ATGGATGATCGAGAGGATCTGGTGTACCAGGCGAAGCTGGCCGAGCAGGCTGAGCGATACGACGAAATGGTGGAGTCAATGAAGAAAGTAGCAGGGATGGATGTGGAGCTGACAGTTGAAGAAAGAAACCTCCTATCTGTTGCATATAAGAATGTGATTGGAGCTAGAAGAGCCTCCTGGAGAATAATCAGCAGCATTGAACAGAAAGAAGAAAACAAGGGAGGAGAAGACAAGCTAAAAATGATTCGGGAATATCGGCAAATGGTTGAGACTGAGCTAAAGTTAATCTGTTGTGACATTCTGGATGTACTGGACAAACACCTCATTCCAGCAGCTAACACTGGCGAGTCCAAGGTTTTCTATTATAAAATGAAAGGGGACTACCACAGGTATCTGGCAGAATTTGCCACAGGAAACGACAGGAAGGAGGCTGCGGAGAACAGCCTAGTGGCTTATAAAGCTGCTAGTGATATTGCAATGACAGAACTTCCACCAACGCATCCTATTCGCTTAGGTCTTGCTCTCAATTTTTCCGTATTCTACTACGAAATTCTTAATTCCCCTGACCGTGCCTGCAGGTTGGCAAAAGCAGCTTTTGATGATGCAATTGCAGAACTGGATACGCTGAGTGAAGAAAGCTATAAGGACTCTACACTTATCATGCAGTTGTTACGTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGTGCTGGGACCGGGCGTGACCGCGAACCCTGGCACCTCCCTGTCTGTGTTCACGGCTCTGCCCTTCACCACACCCGCTCCCGGCCCAGCACACGGGCCGCTCCTTGTGACTGCAGGGGCTCCTCCAGGCGGCCCTCTGGTGCTGTCTACCTTCCCCAGCACACCTCTGGTGACAGAACAGGATGGCTGCGGCCCGAGTGGGGCCGGGGCTTCCAACGTCTTTGTCCAGATGAGGACAGAGGTGGGGCCTGTGAAGGCCGCTCAGGCGCAGACCTTGGTCCTAACTCAGGCCCCCCTCGTCTGGCAGGCTCCAGGCGCCCTCTGCGGAGGTGTTGTGTGTCCACCTCCCCTACTCCTGGCAGCTGCTCCTGTGGTGCCTGTTATGGCTGCCCAGGTGGTTGGGGGCACCCAGGCCTGTGAGGGAGGCTGGTCCCAGGGCCTTCCTCTTCCACCACCACCACCACCGGCTGCCCAGCTGCCCCCCATTGTGTCCCAAGGGAATGCTGGGCCATGGCCACAAGGGGCTCATGGAGAGAGCAGCCTGGCTTCCTCCCAGGCCAAGGCCCCGCCAGATGACTCCTGTAACCCCAGGAGTGTCTATGAGAACTTCCGACTCTGGCAGCACTACAAGCCCCTGGCCCGGAGGCACCTTCCCCAGAGTCCTGACACCGAAGCGCTTTCGTGCTTCCTCATCCCAGTTCTCCGATCGCTGGCCCGGCGGAAGCCCACCATGACCCTGGAGGAGGGACTGTGGCGGGCCATGCGGGAATGGCAGCACACGAGCAACTTTGACCGGATGATCTTCTACGAGATGGCGGAAAAGTTCCTGGAGTTTGAGGCTGAGGAGGAGATGCAGATTCAGAAATCGCAATGGATGAAGGGGCCCCAGTGCCTGCCTCCTCCAGCCACACCGAGGCTTGAACCTCGAGGACCCCCGGCCCCTGAGGTGGTCAAGCAGCCAGTGTACCTTCCCAGCAAGGCCGGCCCCAAGGCCCCGACTGCCTGCCTGCCACCACCCAGGCCCCAGAGGCCAGTGACCAAGGCCCGCCGGCCACCACCCCGGCCCCACCGGCGAGCAGAGACCAAGGCCCGCCTGCCACCACCCAGGCCCCAGAGACCAGCAGAGACCAAGGTCCCTGAGGAGATCCCCCCAGAAGTGGTGCAGGAGTATGTGGACATCATGGAGGAGCTGCTGGGGCCTTCCCTCGGGGCCACGGGGGAGCCCGAGAAACAACGGGAAGAGGGCAAAGTGAAGCAGCCACAGGAAGAGGACTGGACGCCCCCAGACCCGGGCCTCCTGAGCTACATTGACAAGCTGTGTTCCCAGAAAGACTTCGTCACCAAGGTGGAGGCCGTCATTCATCCCCAATTCCTGGAAGAATTGCTTTCCCCAGATCCACAGATGGATTTCTTGGCCCTAAGCCAGGACCTGGAGCAGGAGGAAGGACTCACCCTTGCCCAGCTAGTGGAGAAGCGCCTCCCACCCTTGAAGGAGAAACAGCATGCGAGGGCAGCCCCTAGTCGTGGCACAGCCCGGTTGGACTCAAGTTCTTCTAAGTTTGCAGCTGGCCAAGGAGCAGAGAGAGACGTCCCTGACCCCCAACAAGGGGTTGGCATGGAAACCTGCCCACCCCAGATGACTGCCCGGGACTCTCAGGGACGAGGCAGAGCACACACTGGCATGGCCAGGTCCGAAGACTCTGTTGTGCTTTTGGGATGTCAGGATTCCCCTGGGCTGAGGGCTGCCTGGCCAACCTCTCCTCCCCAGGACCACAGACCCACCTGCCCTGGCGTGGGTACCAAGGATGCCTTGGATCTCCCTGGAGGGTCTCCTGTCAGGGAGTCACATGGGCTGGCTCAGGGGTCAAGTGAGGAGGAGGAACTCCCCAGCCTGGCCTTCCTCTTGGGTTCCCAGCACAAGCTTCTGCCCTGGTGGCTACCCCAGAGCCCTGTCCCTGCCTCGGGCCTTCTCAGCCCAGAAAAGTGGGGACCCCAGGGAACTCATCAGTCCCCATCTGCTGAGAGAAGAGGCCTCAACCTAGCACCTTCTCCTGCCAACAAGGCCAAGAAGCGACCTCTCTTTGGAAGCCTGTCCCCTGCTGAAAAGACACCCTACCCAGGGCCTGGGCTCAGGGTCTCTGGGGAGCAATCCCTGACTTGGGGGCTGGGTGGCCCCTCACAGTCTCAAAAGAGAAAGGGTGACCCCTTGGTCTCCAGGAAGGAGAAGAAGCAGCATTGTAGCCAGT AG(Amino acid sequence of YWHAE-FAM22A fusion protein) SEQ ID NO: 3MDDREDLVYQAKLAEQAERYDEMVESMKKVAGMDVELTVEERNLLSVAYKNVIGARRASWRIISSIEQKEENKGGEDKLKMIREYRQMVETELKLICCDILDVLDKHLIPAANTGESKVFYYKMKGDYHRYLAEFATGNDRKEAAENSLVAYKAASDIAMTELPPTHPIRLGLALNESVFYYEILNSPDRACRLAKAAFDDAIAELDTLSEESYKDSTLIMQLLRDNLTLWTSDMQGDAYPALGPGVTANPGTSLSVFTALPFTTPAPGPAHGPLLVTAGAPPGGPLVLSTLPSTPLVTEQDGCGPSGAGASNVFVQMRTEVGPVKAAQAQTLVLTQaAPLVWQAPGALCGGVVCPPPLLLAAAPVVPVMAAQVVGGTQACEGGWSQGLPLPPPPPPAAQLPPIVSQGNAGPWPQGAHGEGSLASSQAKAPPDDSCNPRSVYENFRLWQHYKPLARRHLPQSPDTEALSCFLIPVLRSLARRKPTMTLEEGLWRAMREWQHTSNFDRMIFYEMAEKFLEFEAEEEMQIQKSQWMKGPQCLPPPATPRLEPRGPPAPEVVKQPVYLPSKAGPKAPTACLPPPRPQRPVTKARRPPPRPHRRAETKARLPPPRPQRPAETKVPEEIPPEVVQEYVDIMEELLGPSLGATGEPEKQREEGEVKQPQEEDWTPPDPGLLSYTDKLCSQKDFVTKVEAVIHPQFLEELLSPDPQMDFLALSQELEQEEGLTLAQLVEKRLLPLKEKQHARAAPSRGTARLDSSSSKFAAGQGAERDVPVPQQGVGMETCPPQTTARDSQGRGRAHTGMARSKDSVVLLGCQDSPGLRAARPTSPPQDHRPTCPGVGTKDALDLPGGSPVRESHGLAQGSSEEEELPSLAFLLGSQHKLLPWWLPQSPVPASGLLSPEKWGPQGTHQFPSAERRGLNLAPSPANKAKKRPLFGSLSPAEKTPHPGPGLRVSGEQSLTWGLGGPSQSQKRKGDPLVSRKEKKQRCSQ(Amino acid sequence of YWHAE-FAM22B fusion protein) SEQ ID NO: 4MDDREDLVYQAKLAEQAERYDEMVESMKKVAGMDVELTVEERNLLSVAYKNVIGARRASWRIISSIEQKEENKGGEDKLKMIREYRQMVETELKLICCDILDVLDKHLIPAANTGESKVFYYKMKGDYHRYLAEFATGNDRKEAAENSLVAYKAASDIAMTELPPTHPIRLGLALNFSVFYYEILNSPDRACRLAKAAFDDAIAELDTLSEESYKDSTLIMQLLRDNLTLWTSDMQGDAYPVLGPGVTANPGTSLSVFTALPFTTPAPGPAHGPLLVTAGAPPGGPLVLSTFPSTPLVTEQDGCGPSGAGASNVFVQMRTEVGPVKAAQAQTLVLTQAPLVWQAPGALCGGVVCPPPLLLAAAPVVPVMAAQVVGGTQACEGGWSQGLPLPPPPPPAAQLPPIVSQGNAGPWPQGAHGESSLASSQAKAPPDDSCNPRSVYENFRLWQHYKPLARRHLPQSPDTEALSCFLIPVLRSLARRKPTMTLEEGLWRAMREWQHTSNFDRMIFYEMAEKFLEFEAEEEMQIQKSQWMKGPQCLPPPATPRLEPRGPPAPEVVKQPVYLPSKAGPKAPTACLPPPRPQRPVTKARRPPPRPHRRAETKARLPPPRPQRPAETKVPEEIPPEVVQEYVDIMEELLGPSLGATGEPEKQREEGKVKQPQEEDWTPPDPGLLSYIDKLCSQKDFVTKVEAVIHPQFLEELLSPDPQMDFLALSQDLEQEEGLTLAQLVEKRLPPLKEKQHARAAPSRGTARLDSSSSKFAAGQGAERDVPDPQQGVGMETCPPQMTARDSQGRGRAHTGMARSEDSVVLLGCQDSPGLRAAWPTSPPQDHRPTCPGVGTKDALDLPGGSPVRESHGLAQGSSEEEELPSLAFLLGSQHKLLPWWLPQSPVPASGLLSPEKWGPQGTHQSPSAERRGLNLAPSPANKAKKRPLFGSLSPAEKTPYPGPGLRVSGEQSLTWGLGGPSQSQKRKGDPLVSRKEKKQHCSQ SEQ ID NO: 6:TGTTACGTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCAT SEQ ID NO: 7:TGTTACGTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCAT SEQ ID NO: 8:GTTACGTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATA SEQ ID NO: 9:GTTACGTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATA SEQ ID NO: 10:GTTACGTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATA SEQ ID NO: 11:TTACGTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATAC SEQ ID NO: 12:TTACGTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATAC SEQ ID NO: 13:TACGTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACC SEQ ID NO: 14:TACGTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACC SEQ ID NO: 15:ACGTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCC SEQ ID NO: 16:CGTGATAATCTGACACTATGGACTTCAGACATGCAAGGTGACGCATACCCA SEQ ID NO: 17:GTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAG SEQ ID NO: 18:GTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAG SEQ ID NO: 19:GTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAG SEQ ID NO: 20:GTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAG SEQ ID NO: 21:GTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAG SEQ ID NO: 22:GTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAG SEQ ID NO: 23:GTGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAG SEQ ID NO: 24:TGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGC SEQ ID NO: 25:TGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGC SEQ ID NO: 26:TGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGC SEQ ID NO: 27:TGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGC SEQ ID NO: 28:TGATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGC SEQ ID NO: 29:GATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCG SEQ ID NO: 30:GATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCG SEQ ID NO: 31:ATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGC SEQ ID NO: 32:ATAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGC SEQ ID NO: 33:TAATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCT SEQ ID NO: 34:AATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATAGCCAGCGCTG SEQ ID NO: 35:AATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTG SEQ ID NO: 36:ATCTGGCACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGG SEQ ID NO: 37:ATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGG SEQ ID NO: 38:ATCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGG SEQ ID NO: 39:TCTGACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGG SEQ ID NO: 40:GACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACC SEQ ID NO: 41:GACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACC SEQ ID NO: 42:GACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACC SEQ ID NO: 43:ACACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCG SEQ ID NO: 44:CACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGG SEQ ID NO: 45:CACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGG SEQ ID NO: 46:ACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGG SEQ ID NO: 47:ACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGG SEQ ID NO: 48:ACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGG SEQ ID NO: 49:ACTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGG SEQ ID NO: 50:CTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGC SEQ ID NO: 51:CTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGC SEQ ID NO: 52:CTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGC SEQ ID NO: 53:CTATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGC SEQ ID NO: 54:TATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCG SEQ ID NO: 55:ATGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGT SEQ ID NO: 56:TGGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTG SEQ ID NO: 57:GGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGA SEQ ID NO: 58:GGACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGA SEQ ID NO: 59:ACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACC SEQ ID NO: 60:ACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACC SEQ ID NO: 61:ACTTCAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACC SEQ ID NO: 62:CTTCAGACATGCACGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCG SEQ ID NO: 63:CAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGA SEQ ID NO: 64:CAGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGA SEQ ID NO: 65:AGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAA SEQ ID NO: 66:AGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAA SEQ ID NO: 67:AGACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAA SEQ ID NO: 68:GACATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAAC SEQ ID NO: 69:ATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAACCCT SEQ ID NO: 70:ATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAACCCT SEQ ID NO: 71:ATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAACCCT SEQ ID NO: 72:ATGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAACCCT SEQ ID NO: 73:TGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAACCCTG SEQ ID NO: 74:TGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAACCCTG SEQ ID NO: 75:TGCAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAACCCTG SEQ ID NO: 76:CAGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAACCCTGGC SEQ ID NO: 77:AGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAACCCTGGCA SEQ ID NO: 78:AGGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAACCCTGGCA SEQ ID NO: 79:AGGGTGACCCATACCCAGCGCTGGGACCGGGCGCGACCGCGAACCCTGGCA SEQ ID NO: 80:GGGTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAACCCTGGCAC SEQ ID NO: 81:GTGACGCATACCCAGCGCTGGGACCGGGCGTGACCGCGAACCCTGGCACCT SEQ ID NO: 81:5′-AGAGGCTGAGAGAGTCGGAGACA CTA-3′ SEQ ID NO: 82:5′-TATGGATGATCGAGAGGATCTGGTG-3′ SEQ ID NO: 83:5′-CAGAACTGGATACGCTGAGT GAAGAA-3′ SEQ ID NO: 84:5′-CTCATAGACACTCCTGG GGTTACAGG-3′ SEQ ID NO: 85:5′-TCTTGCTGGGCCTTAGCTTTG-3′ SEQ ID NO: 86: 5′-TATGTTCCAGGAACCTGTTTA-3′SEQ ID NO: 87: 5′-AAGUUCAGGUCGAUAUGUGCA-3′

All patents and other publications identified in the specification andexamples are expressly incorporated herein by reference for allpurposes. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Further, to the extent not alreadyindicated, it will be understood by those of ordinary skill in the artthat any one of the various embodiments herein described and illustratedcan be further modified to incorporate features shown in any of theother embodiments disclosed herein.

What is claimed is:
 1. An assay for selecting a treatment regimen for asubject with endometrial stromal sarcoma, the assay comprising a step ofdetecting in a biological sample taken from the subject the presence ofa YWHAE-FAM22 fusion protein or a nucleic acid encoding the same,wherein if at least one of the fusion protein or the nucleic acid isdetected, then selecting, and administering, a treatment regimencomprising an effective amount of an anti-cancer agent to the subject.2. The assay of claim 1, wherein the nucleic acid comprises thenucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or the fusionprotein comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:4.
 3. The assay of claim 1, wherein the sample is selected from thegroup consisting of blood, urine, plasma, tissue, cell, and anycombinations thereof.
 4. The assay of claim 1, wherein method comprisescontacting the sample with a first reagent that binds with the nucleicacid or the fusion protein.
 5. The assay of claim 4, wherein the firstreagent is selected from the group consisting of a nucleic acid, anantibody, a small molecule, a polypeptide, a peptide, a lipid, an oligo-or poly-saccharide, and any combinations thereof.
 6. The assay of claim4, wherein the first reagent further comprises a label to produce asignal so as to detect presence of the nucleic acid or the fusionprotein in the sample.
 7. The assay of any of 4, wherein the firstreagent is covalently or non-covalently linked to a solid support. 8.The assay of claim 4, wherein the method further comprises contactingthe sample with a second reagent, wherein the second reagent binds withthe first reagent, the nucleic acid, or the fusion protein.
 9. The assayof claim 8, wherein the first reagent is selected from the groupconsisting of a nucleic acid, an antibody, a small molecule, apolypeptide, a peptide, a lipid, an oligo- or poly-saccharide, and anycombinations thereof.
 10. The assay of claim 8, wherein the secondreagent is covalently or non-covalently linked to a solid support. 11.The assay of claim 8, wherein the second reagent further comprises alabel to produce a signal so as to detect presence of the first reagentbound to the nucleic acid or the fusion protein in the isolated sample.12. The assay of claim 1, wherein the assay comprises subjecting thebiological from the subject to: (i) at least one protein detection assayadapted to determine the presence of a YWHAE-FAM22 fusion protein; or,(ii) at least one nucleic acid sequence detection assay adapted todetermine the presence of a nucleic acid encoding the YWHAE-FAM22 fusionprotein.